Compositions and methods for treating and preventing pathologies including cancer

ABSTRACT

Compositions and methods of treating various disorders by administering a therapeutically effective amount of phenylacetate or pharmaceutically acceptable derivatives thereof or derivatives thereof alone or in combination or in conjunction with other therapeutic agents including retinoids, hydroxyurea, and flavonoids. Intravesicle methods of treatment of cancers phenylacetate. Pharmacologically-acceptable salts alone or in combinations and methods of preventing AIDS and malignant conditions, and inducing cell differentiation are also aspects of this invention. A product as a combined preparation of phenylacetate and a retinoid, hydroxyurea, or flavonid (or other mevalonate pathway inhibitor) for simultaneous, separate, or sequential use in treating a neoplastic condition in a subject. Methods of modulating lipid metabolism and/or reducing serum triglycerides in a subject using phenylacetate.

This application is a divisional of U.S. application Ser. No.08/207,521, filed Mar. 7, 1994, pending, which is (1) acontinuation-in-part of Applicant's Ser. No. 08/135,661, filed Oct. 12,1993, pending and also is (2) a continuation-in-part of Applicant's U.S.Ser. No. 07/779,744, filed Oct. 21, 1991, and now abandoned, thecontents of all of which are hereby incorporated by this reference.

I. FIELD OF THE INVENTION

This invention relates to methods of using phenylacetic acid and itspharmaceutically acceptable derivatives to treat and prevent pathologiesand to modulate cellular activities. In particular, this inventionrelates to A) phenylacetate and its derivatives in cancer prevention andmaintenance therapy, B) phenylacetate and its derivatives in thetreatment and prevention of AIDS, C) induction of fetal hemoglobinsynthesis in β-chain hemoglobinopathy by phenylacetate and itsderivatives, D) use of phenylacetic acid and its derivatives in woundhealing, E) use of phenylacetic acid and its derivatives in treatment ofdiseases associated with interleukin-6, F) use of phenylacetic acid andits derivatives in the treatment of AIDS-associated CNS dysfunction, G)use of phenylacetic acid and its derivatives to enhanceimmunosurveillance, H) methods of monitoring the dosage level ofphenylacetic acid and its derivatives in a patient and/or the patientresponse to these drugs, I) the activation of the PPAR by phenylaceticacid and its derivatives, J) use of phenylacetic acid and itsderivatives in treatment of cancers having a multiple-drug resistantphenotype, K) phenylacetic acid and its derivatives, correlation betweenpotency and lipophilicity, L) phenylacetic acid and its derivatives insynergistic combination with lovastatin for the treatment and preventionof cancers such as malignant gliomas or other CNS tumors, M)phenylacetic acid and its derivatives in synergistic combination withretinoic acid for the treatment and prevention of cancers such as thoseinvolving neuroblastoma cells, N) phenylacetic acid and its derivativesfor the treatment and prevention of cancers and other differentiationdisorders such as those involving malignant melanoma or otherneuroectodermal tumors, O) phenylacetic acid and its derivatives insynergistic combination with hydroxyurea (HU) for the treatment andprevention of cancers such as prostate cancer, P) phenylacetic acid andits derivatives for the treatment and prevention of cancers involvingmedulloblastoma and astrocytoma derived cells, Q) phenylacetic acid andits derivatives in human studies relating to treatments with PA and PB,R) phenylacetic acid and its derivatives in methods of altering lipidmetabolism, including reducing serum triglycerides, and S) methods ofadministering phenylacetic acid and its derivatives.

II. BACKGROUND OF THE INVENTION

Phenylacetic acid (PAA) is a protein decomposition product foundthroughout the phylogenetic spectrum, ranging from bacteria to man.Highly conserved in evolution, PAA may play a fundamental role in growthcontrol and differentiation. In plants, PAA serves as a growth hormone(auxin) promoting cell proliferation and enlargement at low doses (10⁻⁵-10⁻⁷ M), while inhibiting growth at higher concentrations. The effecton animal and human cells is less well characterized. In humans, PAA isknown to conjugate glutamine with subsequent renal excretion ofphenylacetylglutamine (PAG). The latter, leading to waste nitrogenexcretion, has been the basis for using PAA or preferably its saltsodium phenylacetate (NaPA, also referenced herein as that activeanionic meoity, phenylacetate or "PA") in the treatment ofhyperammonemia associated with inborn errors of ureagenesis. Clinicalexperience indicates that acute or long-term treatment with high NaPAdoses is well tolerated, essentially free of adverse effects, andeffective in removing excess glutamine. Brusilow, S. W., Horwich, A. L.Urea cycle enzymes. Metabolic Basis of Inherited Diseases, Vol.6:629-633 (1989)!. These characteristics should be of value intreatments of cancer and prevention of cancer, treatments which inhibitvirus replication and treatments of severe beta-chainhemoglobinopathies.

Glutamine is the major nitrogen source for nucleic acid and proteinsynthesis, and a substrate for energy in rapidly dividing normal andtumor cells. Compared with normal tissues, most tumors, due to decreasedsynthesis of glutamine along with accelerated utilization andcatabolism, operate at limiting levels of glutamine availability, andconsequently are sensitive to further glutamine depletion. Consideringthe imbalance in glutamine metabolism in tumor cells and the ability ofPAA to remove glutamine, PAA has been proposed as a potential antitumoragent; however, no data has previously been provided to substantiatethis proposal. Neish, W. J. P. "Phenylacetic Acid as a PotentialTherapeutic Agent for the Treatment of Human Cancer", Experentia, Vol.27, pp. 860-861 (1971)!.

Despite these efforts to fight cancer, many malignant diseases that areof interest in this application continue to present major challenges toclinical oncology. Prostate cancer, for example, is the second mostcommon cause of cancer deaths in men. Current treatment protocols relyprimarily on hormonal manipulations. However, in spite of initial highresponse rates, patients often develop hormone-refractory tumors,leading to rapid disease progression with poor prognosis. Overall, theresults of cytotoxic chemotherapy have been disappointing, indicating along felt need for new approaches to treatment of advanced prostaticcancer. Other diseases resulting from abnormal cell replication, forexample metastatic melanomas, brain tumors of glial origin (e.g.,astrocytomas), and lung adenocarcinoma, are also highly aggressivemalignancies with poor prognosis. The incidence of melanoma and lungadenocarcinoma has been increasing significantly in recent years.Surgical treatments of brain tumors often fail to remove all tumortissues, resulting in recurrences. Systemic chemotherapy is hindered byblood barriers. Therefore, there is an urgent need for new approaches tothe treatment of human malignancies including advanced prostatic cancer,melanoma, brain tumors.

The development of the methods and pharmaceuticals of the presentinvention was guided by the hypothesis that metabolic traits thatdistinguish tumors from normal cells could potentially serve as targetsfor therapeutic intervention. For instance, tumor cells show uniquerequirements for specific amino acids such as glutamine. Thus, glutaminemay be a desired choice because of its major contribution to energymetabolism and to synthesis of purines, pyrimidines, and proteins. Alongthis line, promising antineoplastic activities have been demonstratedwith glutamine-depleting enzymes such as glutaminase, and variousglutamine antimetabolites. Unfortunately, the clinical usefulness ofthese drugs has been limited by unacceptable toxicities. Consequently,the present invention focuses on PAA, a plasma component known toconjugate glutamine in vivo, and the pharmaceutically acceptablederivatives of PAA.

In addition to its ability to bind gluatamine to form glutaminephenylacetate, phenylacetic acid (PAA) can induce tumor cells to undergodifferentiation. (See examples 1-5, 7-9, 11-13, and 16 herein).Differentiation therapy is a known, desirable approach for cancerintervention. The underlying hypothesis is that neoplastictransformation results from defects in cellular differentiation.Inducing tumor cells to differentiate would prevent tumor progressionand bring about reversal of malignancy. Several differentiation agentsare known, but their clinical applications have been hindered byunacceptable toxicities and/or deleterious side effects.

The utility of PAA and its derivatives is more fully delineated in theabove-referenced copending applications. As discussed in theseapplications, PAA is a nontoxic differentiation enhancer and hasantitumor activity in laboratory models and in man. Preclinical studiesindicate that phenylacetate and related aromatic fatty acids inducecytostasis and promote maturation of various human malignant cells,including hormone-refractory prostatic carcinoma and glioblastoma. Themarked changes in tumor biology are associated with alterations in theexpression of genes implicated in tumor growth, invasion, angiogenesis,and immunogenicity. PAA and its analogs appear to share severalmechanisms of action, including (a) regulation of gene expressionthrough activation of a nuclear receptor; and (b) inhibition of themevalonate pathway and protein isoprenylation. Thus, PAA appearsparticularly suited in the treatment of various neoplastic conditions.

One such neoplastic condition treatable by NaPA is neuroblastoma. As amalignant tumor of childhood, neuroblastoma has proven to be fascinatingfrom a biological as well as clinical veiwpoint. This cancer has thehighest rate of spontaneous differentiation of all malignancies andseveral agents have been reported to induce maturation of neuroblastomainto a variety of cells sharing a neural-crest lineage (Evans, A. E.,Chatten, J., D'Angio, G. J., Gerson, J. M., Robinson, J., and Schnaufer,L. A review of 17 IV-S neuroblastoma patients at the Children's Hospitalof Philadelphia. Cancer, 45:833-839, 1980; Abemayor, E., and Sidell, N.Human neuroblastoma cell lines as models for the in vitro study ofneoplastic an neuronal cell differentiation. Environ. Health Perspect.,80:3-15, 1989). Among the compounds that have been explored asdifferentiating agents, retinoic acid (RA) (Sidell, N., Altman, A.,Haussler, M. R., and Seeger, R. C. Effects of retinoic acid (RA) on thegrowth and phenotypic expression of several human neuroblastoma celllines. Expl. Cell Res., 148:21-30, 1983) was shown to be a potentcompound for promoting the differentiation of a variety of humanneuroblastoma cell lines (Abemayor, E., and Sidell, N. Humanneuroblastoma cell lines as models for the in vitro study of neoplastican neuronal cell differentiation. Environ. Health Perspect., 80:3-15,1989; Sidell, N., Altman, A., Haussler, M. R., and Seeger, R. C. Effectsof retinoic acid (RA) on the growth and phenotypic expression of severalhuman neuroblastoma cell lines. Expl. Cell Res., 148:21-30, 1983;Thiele, C. T., Reynolds, C. P., and Israel, M. A. Decreased expressionof N-myc precedes retinoic acid-induced morphological differentiation ofhuman neuroblastoma. Nature, 313:404-406, 1985); however, to date RA hasdemonstrated only limited clinical effectiveness in this disease(Finklestein, J. Z., Krailo, M. D., Lenarsky, C., Ladisch, S., Blair, G.K., Reynolds, C. P., Sitary, A. L., and Hammond, G. D., 13-cis-retinoicacid (NSC 122758) in the treatment of children with metastaticneuroblastoma unresponsive to conventional chemotherapy: Report from theChildren's Cancer Study Group. Med. Ped. Oncol., 20:307-311, 1992). Inpursuit of increasing the efficacy of RA-induced differentiation ofhuman neuroblastoma, a number of other compounds and biological responsemodifiers, such as cAMP-elevating agents and interferons, can potentiatethe retinoid activity as well as render resistant populations sensitiveto RA treatment (Lando, M., Abemayor, E., Verity, M. A., and Sidell, N.Modulation of intracellular cyclic AMP levels and the differentiationresponse of human neuroblastoma cells. Cancer Res., 50:722-727, 1990;Wuarin, L., Verity, M. A., and Sidell, N. Effects of gamma-interferonand its interaction with retinoic acid on human neuroblastoma cells.Int. J. Cancer, 48:136-144, 1991). Some of these combination treatmentsare now being evaluated clinically or proposed for the treatment ofneuroblastoma and other malignancies (Smith, M. A., Parkinson, D. R.,Cheson, B. D., and Friedman, M. A. Retinoids in cancer therapy. J. Clin.Oncol., 10:839-864, 1992). However, there exists a need for moreeffective combination treatments for the treatment of neuroblastoma andother similar cancers and pathologies.

Another neoplastic condition which heretofore has been difficult totreat is malignant glioma. Malignant gliomas are highly dependent on themevalonate (MVA) pathway for the synthesis of sterols and isoprenoidscritical to cell replication (Fumagalli, R., Grossi, E., Paoletti, P.and Paolette, R. Studies on lipids in brain tumors. I. Occurrence andsignificance of sterol precursors of cholesterol in human brain tumors.J. Neurochem. 11:561-565, 1964; Kandutsch, A. A. and Saucier, S. E.Regulation of sterol synthesis in developing brains of normal and gimpymice. Arch. Biochem. Biophys. 135:201-208, 1969; Grossi, E., Paoletti,P. and Paoletti, R. An analysis of brain cholesterol and fatty acidbiosynthesis. Arch. Int. Physiol. Biochem. 66:564-572, 1958; Azarnoff,D. L., Curran, G. L. and Williamson, W. P. Incorporation of acetate-1-¹⁴C into cholesterol by human intracranial tumors in vitro. J. Nat. CancerInst. 21:1109-1115, 1958; Rudling, M. J., Angelin, B., Peterson, C. O.and Collins, V. P. Low density lipoprotein receptor activity in humanintracranial tumors and its relation to cholesterol requirement. CancerRes. 50 (suppl):483-487, 1990). Targeting MVA synthesis and/orutilization would be expected to inhibit tumor growth without damagingnormal brain tissues, in which the MVA pathway is minimally active. Twoenzymes control the rate limiting steps of the MVA pathway ofcholesterol synthesis: (a) 3-hydroxy-3-methylglutaryl coenzyme A(HMG-CoA) reductase catalyzes the synthesis of MVA from acetyl-CoA; and,(b) MVA-pyrophosphate (MVA-PP) decarboxylase controls MVA utilizationand, consequently, the post-translational processing and function ofintracellular signalling proteins (Goldstein, J. L. and Brown, M. S.Regulation of the mevalonate pathway. Nature. 343:425-430, 1990;Marshall, C. J. Protein prenylation: A mediator of protein-proteininteractions. Science. 259:1865-1866, 1993). Therefore, it is highlydesirable for the treatment of malignant gliomas or other similarcancers and pathologies to find a treatment capable of inhibiting thesetwo steps of the MVA pathway.

A further neoplastic condition which has been difficult to treat ismalignant melanoma. Disseminated malignant melanoma is characterized bya high mortality rate and resistance to conventional therapies (FerdyLejeune, Jean Bauer, Serge Leyvraz, Danielle Lienard (1993):Disseminated melanoma, preclinical therapeutic studies, clinical trial,and patient treatment. Current Opinion in Oncology 5:390-396).Differentiation therapy may provide an alternative for treatment ofcancers that do not or poorly respond to cytotoxic chemotherapy(Kelloff, G. J., Boone, C. W., Malone, E. F., Steele, V. E. (1992):Chemoprevention clinical trials. Mutation Res. 267:291-295). Severaldifferentiation inducers are capable of altering the phenotype ofmelanoma cells in vitro. These include retinoids, butyrate, dibutyryladenosine 3':5'-cyclic monophosphate (dbc AMP), 5-Azacytidine,interferons, hexamethylene bisacetamide (HMBA), dimethyl formamide(DMF), dimethyl sulfoxide (DMSO), 12-tetradecanoylphorbol-13 acetate(TPA) (Mary J. Hendrix, Rebecca W. Wood, et al. (1990): Retinoic acidinhibition of human melanoma cell invasion through a reconstitutedbasement membrane and its relation to decreases in the expression ofproteolytic enzymes and motility. Cancer Research 50:4121-4130; LuaraGiffre, Magali Schreyer, Jean-pierre Mach, Stefan Carrel (1988): CyclicAMP induces differentiation in vitro of human melanoma cells. Cancer61:1132-1141; Jardena Nordenberg, Lina Wasserman, Einat Beery, DoronAloni, Hagit Malik, Kurt H. Stenzel, Abraham Novogrodsky (1986): Growthinhibition of murine melanoma by butyric acid and dimethylsulfoxide.Experimental Cell Research 162:77-85; Eliezer Huberman, Carol Heckman,Rober Langenbach (1979): Stimulation of differentiation functions inhuman melanoma cells by tumor-promoting agents and dimethyl sulfoxide.Cancer Research 39:2618-2624; Claus Garbe, Konstantin Krasagakis (1993):Effects of interferons and cytokines on melanoma cells. J. Invest.Dermatol. 100:239S-244S). Unfortunately, clinical applications of theseagents are limited by unacceptable toxicities, concern regardingpotential carcinogensis, or an inability to achieve and sustaineffective plasma concentrations. Therefore, there exists a need for anontoxic, clinically effective treatments for malignant melanomas orother similar cancers, pathologies or differentiation disorders.

Hydroxyurea, a ribonucleotide reductase inhibitor, is a simple chemicalcompound (CH₄ N₂ O₂, MW 76.05) that was initially synthesized in thelate 1800's (Calabresi P., Chabner B. A. Antineoplastic Agents. In:Gilman A. G., Rall T. W., Nies A. S., Taylor P., eds. ThePharmacological Basis of Therapeutics. New York: McGraw Hill1990:1251-2). It was later found to produce leukopenia in laboratoryanimals and subsequently was tested as an antineoplastic agent(Rosenthal F., Wislicki L., Kollek L. Ueber die beziehungen vonschwersten blutgiften zu abbauprodukten des ewweisses. Beitrag zumentstehungsmechanismus der perniziosen. Anamie. Klin. Wschr.1928;7:972). At present, the primary clinical role of hydroxyurea is inthe treatment of myeloproliferative disorders. It is now considered thepreferred initial therapy for chronic myelogenous leukemia (Donehower R.C. Hydroxyurea. In: Chabner B. A., Collins J. M., eds CancerChemotherapy, Principles and Practice, Philadelphia: J. B. Lippincott1990:225-33).

Hydroxyurea has been evaluated in a number of solid tumors, including:malignant melanoma, squamous cell carcinoma of the head and neck, renalcell carcinoma, and transitional cell carcinoma of the urothelium(Bloedow C. E. A phase II study of hydroxyurea in adults: miscellaneoustumors. Cancer Chemoother Rep 1964;40-39-41; Ariel I. M. Therapeuticeffects of hydroxyurea: experience with 118 patients with inoperabletumors. Cancer 1970;25:714; Nevinny H., Hall T. C. Chemotherapy withhydroxyurea in renal cell carcinoma. J Clin Pharmacol 1968;88:352-9;Beckloff G. L., Lerner H. J., Cole D. R., et al. Hydroxyurea in bladdercarcinoma. Invest Urol 1967;6:530-4). Initial studies appeared promisingin several of these diseases, but further investigation has not defineda role for hydroxyurea in any of the standard therapy regimens for solidtumors.

Inasmuch as hydroxyurea is an S-phase cell cycle specific agent, it issurprising that several clinical trials of this drug inhormone-refractory metastatic prostate cancer suggested that itpossessed some activity (Lerner H. J., Malloy T. R. Hydroxyurea in stageD carcinoma of prostate. Urol 1977;10,35-8; Kvols L. K., Eagan R. T.,Myers R. P. Evaluation of melphalan, ICRF-159, and hydroxyurea inmetastatic prostate cancer: a preliminary report. Cancer Treat Rep1977;61:311-2; Loening S. A., Scott W. W., deKernion J, et al. AComparison of hydroxyurea, methyl-chloroethyl-cyclohexy-nitrosourea andclylophosphamide in patients with advance carcinoma of the prostate. JUrol 1981;125:812-6; Mundy A. R. A pilot study of hydroxyurea in hormone"escaped" metastatic carcinoma of the prostate. Br J Urol 1982;54:20-5;Stephens R. L., Vaughn C., Lane M., et al. Adriamycin andcyclophosphamide versus hydroxyurea in advanced prostatic cancer. Cancer1984;53:406-10) particularly given (1) the slowly progressive nature ofthe disease and (2) the schedules of drug administration used in thesetrials, e.g., once daily to once every three days. It seemed unlikelythat either the doses or schedules of hydroxyurea administration used inthese trials would capture a significant proportion of tumor cells in asusceptible phase of the cell cycle. Table 30 summarizes the reportedclinical data regarding hydroxyurea's activity in hormone-refractoryprostate cancer. The overall objective response rate is 23% and thefrequency of subjective improvement is 36% (Lerner H. J., Malloy T. R.Hydroxyurea in stage D carcinoma of prostate. Urol 1977;10,35-8; KvolsL. K., Eagan R. T., Myers R. P. Evaluation of melphalan, ICRF-159, andhydroxyurea in metastatic prostate cancer: a preliminary report. CancerTreat Rep 1977;61:311-2; Loening S. A., Scott W. W., deKernion J, et al.A Comparison of hydroxyurea, methyl-chloroethyl-cyclohexy-nitrosoureaand clylophosphamide in patients with advance carcinoma of the prostate.J Urol 1981;125:812-6; Mundy A. R. A pilot study of hydroxyurea inhormone "escaped" metastatic carcinoma of the prostate. Br J Urol1982;54:20-5; Stephens R. L, Vaughn C., Lane M., et al. Adriamycin andcyclophosphamide versus hydroxyurea in advanced prostatic cancer. Cancer1984;53:406-10). Thus, there exists a need for an improved therapy usinghydroxyurea for treatment of prostatic or similar cancers.

Treatment for most primary central nervous system (CNS) tumors has been,to date, unsatisfactory. Chemotherapy, radiation therapy, and surgeryare primarily cytoreductive, aiming to reduce the number of viable tumorcells in the host. While the application of these techniques has beensuccessful in some human malignancies, the use of cytoreductivestrategies for medulloblastoma and malignant astrocytoma has had limitedsuccess because of inaccessibility of the primary tumor, earlydissemination of the malignant cells into the cerebrospinal fluid, lackof effective cytoreductive agents or unacceptable toxicity. Thus, thereexists a need for satisfactory treatments for CNS tumors which overcomethe prior drawbacks of conventional cytoreductive therapy.

In addition, the link between problems with lipid metabolism and heartdisease are now well-accepted. Thus, it is desirable to find treatmentcapable of modulating or altering lipid metabolism in subjects withrelated maladies. In particular treatments and methods for reducingserum triglycerides are highly desirable.

While radiation therapy has been widely used in the management ofneoplastic disease, it is limited by the lack of radiosensitivity ofspecific regions of malignant tumors. Chemical enhancement of tumorsensitivity to radiation has largely been unsuccessful and remains acritical problem in radiotherapy. Thus, there exists a need for improvedmethods involving radiotherapy.

Accordingly, the present invention provides methods and compositions fortreating the above-mentioned and other pathologies with PAA and itspharmaceutically acceptable salts, derivatives, and analogs.

III. SUMMARY OF THE INVENTION

The invention provides a method of treating various pathologies in asubject. The invention also provides for the modulation of variouscellular activities in a subject. The pathologies and cellularactivities are treated and modulated utilizing a compound having theformula: ##STR1## ; wherein R₀ =aryl, phenoxy, substituted aryl orsubstituted phenoxy;

R₁ and R₂ =H, lower alkoxy, lower straight and branched chain alkyl orhalogen;

R₃ and R₄ =H, lower alkoxy, lower straight and branched chain alkyl orhalogen; and

n=an integer from 0 to 2.

Specifically, the invention provides a method of treating or preventingvarious neoplastic conditions. Relatedly, a method of inducingdifferentiation of a cell is provided. The invention also provides amethod of inducing the production of fetal hemoglobin and treatingpathologies associated with abnormal hemoglobin activity or production.

The invention also provides a method of treating or preventing a viralinfection in a subject. Relatedly, the invention provides a method oftreating an AIDS-associated dysfunction of the central nervous system ina subject.

Also provided is a method of modulating the production of IL-6 or TGFαand TGF-β2 both in vitro and in vivo. Typically, IL-6 and TGF-β2 areinhibited while TGFα is induced.

The invention also provides a method of enhancing immunosurveillance andpromoting wound healing in a subject.

Also provided is a method of monitoring the bioavailability of acompound for treatment of a pathology not associated with hemoglobin.The method comprises administering to a subject the compound andmeasuring the level of fetal hemoglobin TGF-β2, IL-6 or TGFα.

A method of treating a neoplastic condition in cells resistant toradiation and chemotherapy is provided. Specifically, multiple drugresistant cells are particularly sensitive to the compounds of thisinvention.

The present invention provides, in several embodiments, combinationswhich inhibit certain key regulatory enzymes. Thus, HMG-CoA reductaseand MVA-PP decarboxylase can be blocked by lovastatin (LOV) andphenylacetate (PA), respectively.

In another embodiment, the present invention overcomes the toxicityproblems with monotreatments with hydroxyurea. Because it wasanticipated that the plasma concentrations of hydroxyurea required forcytotoxicity would result clinically in an unacceptable degree ofmyelosuppression, another objective of the present invention was toevaluate the activity in combination with phenylbutyrate, a relativelynon-toxic differentiating agent.

In other embodiments, the present invention emcompasses the followingsubject matter:

The present invention provides a method of treating a neoplasticcondition in a subject comprising administering a therapeutic amount ofa phenylacetic acid derivative of the formula:

General Structure A: ##STR2## ; wherein R₀ =aryl, phenoxy, substitutedaryl or substituted phenoxy;

R₁ and R₂ =H, lower alkoxy, lower straight and branched chain alkyl orhalogen;

R₃ and R₄ =H, lower alkoxy, lower straight and branched chain alkyl orhalogen; and

n=an integer from 0 to 2; salts thereof; stereoisomers thereof; andmixtures thereof. This general structure is hereinbelow referred to asGeneral Structure A without reference to any particular method orcomposition. The neoplastic conditions treatable by this method includeneuroblastoma, acute promyelocytic leukemia, acute myelodisplasia, acuteglioma, prostate cancer, breast cancer, melanoma, non-small cell lungcancer, medulloblastoma, and Burkitt's lymphoma. The compounds for theabove method (as disclosed in General Structure A), and for any of themethods or compositions disclosed elsewhere herein, specifically includesodium phenylacetate and sodium phenylbutyrate.

Further provided is a method of preventing a neoplastic condition in asubject comprising administering a prophylactic amount of a phenylaceticacid derivative of General Structure A. This method encompasses a methodwhere the compound is administered in combination with ananti-neoplastic agent.

The present invention also provides a method of inducing thedifferentiation of a cell comprising administering to the cell adifferentiation inducing amount of a phenylacetic acid derivative ofGeneral Structure A.

Also included is a method of inducing the production of fetal hemoglobinin a subject comprising administering to the subject a fetal hemoglobininducing amount of a phenylacetic acid derivative of General StructureA.

The present invention includes a method of treating a pathologyassociated with abnormal hemoglobin activity in a subject comprisingadministering to the subject a therapeutic amount of a phenylacetic acidderivative of General Structure A. This method may be used to treat apathology which is anemia. More specifically, the anemia may be selectedfrom the group consisting of sickle cell and beta thalassemia.

Further provided is a method of treating a viral infection in a subjectcomprising administering to the subject a therapeutic amount of aphenylacetic acid derivative of General Structure A. The viral infectiontreated by the above method may be an infection by a retrovirus.Specifically, this method may be used to treat a subject infected by aHuman Immunodeficiency Virus.

The present invention provides a method of preventing a viral infectionin a subject comprising administering to the subject a prophylacticamount of a phenylacetic acid derivative of General Structure A.

In another embodiment, the present invention provides a method ofinhibiting the production of IL-6 in a cell comprising contacting thecell with an IL-6 inhibiting amount of a phenylacetic acid derivative ofGeneral Structure A. This method of inhibiting may be used in a subjecthaving any of the following pathologies: rheumatoid arthritis,Castleman's disease, mesangial proliferation, glomerulonephritis,uveitis, sepsis, automimmunity inflammatory bowel, type I diabetes,vasculitis and a cell differentiation associated skin disorder. Theinhibition, of course, will be sufficient to treat the disorder.

The present invention also provides a method of inducing the productionof TGFα in a cell comprising contacting the cell with a TGFα inducingamount of a phenylacetic acid derivative of General Structure A. Thismethod may be used where the induction is in a wound of a subject andthe induction is sufficient to promote wound healing.

The present invention also provides a method of inhibiting theproduction of TFG-β2 in a cell comprising contacting the cell with aTGF-β2 inhibiting amount of a phenylacetic acid derivative of GeneralStructure A.

Further provided is a method of treating an AIDS-associated dysfunctionof the central nervous system in a subject comprising administering tothe subject a therapeutic amount of a phenylacetic acid derivative ofGeneral Structure A.

Another embodiment of the present invention is a method of enhancingimmunosurveillance in a subject comprising administering to the subjectan immunosurveillance enhancing amount of a phenylacetic acid derivativeof General Structure A.

The present invention also provides a method of monitoring thebioavailability of a compound of General Structure A. This method isapplicable for the treatment of a pathology not associated withhemoglobin and it comprises administering to a subject the compound andmeasuring the level of fetal hemoglobin, an increase in the amount offetal hemoglobin indicating an increased bioavailability of the compoundto treat the pathology and a decrease in the amount of fetal hemoglobinindicating a decrease in the bioavailability of the compound to treatthe pathology. This method is useful for monitoring a pathology which isa neoplastic condition.

The present invention provides a method of promoting the healing of awound in a subject comprising administering to a wound in the subject awould healing amount of a phenylacetic acid derivative of GeneralStructure A.

Further provided is a method of treating a neoplastic condition in asubject resistant to radiation and chemotherapy comprising administeringto said subject a therapeutic amount of a phenylacetic acid derivativeof General Structure A. This method is particularly useful for treatmentof a neoplastic condition exhibiting the multiple drug resistantphenotype.

Thus, the present invention also includes the following embodiments:

A method of treating a neoplastic condition in a subject comprisingadministering a therapeutic amount of a retinoid in combination with atherapeutic amount of a phenylacetic acid derivative of GeneralStructure A.

The present invention also provides a method of treating a neoplasticcondition in a subject comprising administering a therapeutic amount ofan inhibitor of the mevalonate pathway in combination with a therapeuticamount of a phenylacetic acid derivative of General Structure A. Arelated method uses the above steps and further includes the steps ofcontinuously monitoring the subject for rhabdomyolysis-induced myopathyand in the presence of rhabdomyolysis-induced myopathy, administeringubiquinone to the subject.

A further method of the present invention is a method of inhibitingHMG-coA reductase and MVA-PP decarboxylase in a subject with aneoplastic condition, comprising administering a therapeutic amount ofan inhibitor of the mevalonate pathway in combination with a therapeuticamount of a phenylacetic acid derivative of General Structure A. Arelated method also includes the additional steps of continuouslymonitoring the subject for rhabdomyolysis-induced myopathy and in thepresence of rhabdomyolysis-induced myopathy, administering ubiquinone tothe subject.

The present invention also provides a method of treating a neoplasticcondition in a subject, comprising administering a therapeutic amount ofa flavonoid in combination with a therapeutic amount of a phenylaceticacid derivative of General Structure A.

The present invention provides a further method of treating a neoplasticcondition in a subject, comprising administering a therapeutic amount ofhydroxyurea in combination with a therapeutic amount of a phenylaceticacid derivative of General Structure A.

In another embodiment, the present invention provides a method ofmodulating lipid metabolism in a subject, comprising administering atherapeutic amount of a phenylacetic acid derivative of GeneralStructure A. In a related embodiment, the present invention provides amethod of reducing serum triglycerides in a subject, comprisingadministering a therapeutic amount of a phenylacetic acid derivative ofGeneral Structure A.

The present invention further provides a method of locally treating aneoplastic condition of an internal tissue of a subject, comprisingadministering, intravesically, a therapeutic amount of a phenylaceticacid derivative of General Structure A.

The present invention provides a method of sensitizing a subject toradiation therapy, comprising administering a therapeutic amount of aphenylacetic acid derivative of General Structure A.

Also provided is a product for simultaneous, separate, or sequential usein treating a neoplastic condition in a subject, comprising, in separatepreparations, a therapeutic amount of a vastatin and a therapeuticamount of a phenylacetic acid derivative of General Structure A.

A further embodiment of the present invention provides a product forsimultaneous, separate, or sequential use in treating a neoplasticcondition in a subject, comprising, in separate preparations, atherapeutic amount of a retinoid and a therapeutic amount of aphenylacetic acid derivative of General Structure A.

The present invention also provides a product for simultaneous,separate, or sequential use in treating a neoplastic condition in asubject, comprising, in separate preparations, a therapeutic amount ofhydroxyurea and a therapeutic amount of a phenylacetic acid derivativeof General Structure A.

The present invention provides a composition, comprising a therapeuticamount of a vastatin and a therapeutic amount of a phenylacetic acidderivative of General Structure A.

The present invention further provides a composition, comprising atherapeutic amount of a retinoid and a therapeutic amount of aphenylacetic acid derivative of General Structure A.

Finally, the present invention provides a composition, comprising atherapeutic amount of hydroxyurea and a therapeutic amount of aphenylacetic acid derivative of General Structure A.

IV. BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the inhibition of HL-60 leukemia and premalignant 10T1/2cell proliferation by NaPA.

FIG. 2 shows the induction of HL-60 cell differentiation. The number ofNRT positive cells was determined after 4 solid bars! or 7 days hatchedbars! of treatment. NaPA (h), 1.6 mg/ml; NaPA (1), 0.8 mg./ml.4-hydroxyphenylacetate and DAC were used at 1.6 mg./ml. Potentiation byRA 10 nM was comparable to that by IFN gamma 300 IU/ml, and the effectof acivicin 3 μg/ml similar to DON 30 μg/ml. Glutamine Starvation(Gln,>0.06 nM) was as described. Cell viability was over 95% in allcases, except for DON and acivicin (75% and 63%, respectively).

FIG. 3 shows adipocyte conversion in 10T1/2 cultures.

FIG. 4 shows NaPA's ability to invoke growth arrest of humanglioblastoma cello. Dose-dependent inhibition of human glioblastoma cellproliferation by sodium phenylacetate. Growth rates were determined,after 4-5 days of continuous treatment, by an enzymatic assay using 3-4,5-dimethylthiazol-2-yl!-2,5-diphenyltertrazolium bromide and confirmedby cell enumeration with a hemocytometer. Reduction in cell numberparalleled changes in de novo DNA synthesis (not shown).

FIG. 5 shows selective cytostasis induced by phenylacetate (5 mM)combined with glutamine starvation (0.2 mM glutamine, i.e. 2-3 foldbelow the normal plasma levels). The results indicate increasedvulnerability of glioblastoma A172 when compared to actively replicatingnormal human umbilical vein endothelial cells (HUVC). Cell viability wasover 95% in all cases.

FIG. 6 shows that phenylacetate inhibits the mevalonate pathway ofcholesterol synthesis in glioblastoma cells. FIG. 6 shows key steps ofthe MVA pathway discussed in text.

FIG. 7 shows the selective inhibition of cholesterol synthesis frommevalonate in phenylacetate-treated glioblastoma U87 cells, andenzymatic inhibition is of mevalonate decarboxylation in cellhomogenates. For analysis of steroid synthesis, logarithmically growingcells were labeled with tritiated MVA in the presence or absence of 5 mMphenylacetate, and their steroids were separated by silica thin layerchromatography. MVA decarboxylation was measured in cell homogenates.The effect of phenylacetate on cholesterol synthesis and MVAdecarboxylation was selective as, under the experimental conditionsused, total protein and DNA synthesis levels were unaffected.

FIG. 8 shows the effects of phenylacetate on rate of proliferation afterin vitro exposure of 9 L tumor cells to various concentrations ofphenylacetate for 5 days. Significant decline in DNA-synthesis wasobserved. Data are expressed as means±S.D. counts per minute (cpm).

FIG. 9 shows the treatment with phenylacetate from the day ofintracerebral tumor inoculation extended survival compared withtreatment with saline (p<0.0; Mantel-Haenzel test).

FIG. 10 shows the treatment of established tumors with phenylacetateextended survival compared to treatment with saline (p<0.03;Mantel-Haenzel test).

FIG. 11 shows the effect of NaPA on cell proliferation. PC3; DU145;LMCaP; and FS4 cultures were treated with NaPA or PAG for four days.

FIG. 12 shows a chromatogram of phenylacetate (PA) andphenylacetylglutamine (PAG). The peaks at 9.8 and 17.1 minutes representPAG and PA, respectively. serum concentrations of 250 μg/ml in bothinstances.

FIG. 13 shows serum concentrations of PA ( ) and PAG ( ) and plasmaconcentrations of glucamine ( ) following a 150 mg/kg i.v. bolus of PAover 2 hours.

FIG. 14 shows declining phenylacetate concentrations over time duringCIVI (250 mg/kg/day) in one patient, suggestive of clearance induction.

FIG. 15 shows the inhibition of tumor cell invasion by NaPA cellstreated in culture for seven (7) days which were harvested and assayedfor their invasive properties using a modified Boyden Chamber with amatrigel-coated filter. Results were scored six (6) to twenty-four (24)hours later.

FIG. 16 shows a simulation of a q 12 hour PA regimen (200 mg/kg/dose, 1hour infusion) in a pharmacokinetically average patient. For simplicity,induction of clearance was not factored in.

FIG. 17 shows the effect of NaPA on cell growth and differentiation. (∘)Total cell number and () the traction of benzidine-positive cells weredetermined after 4 days of continuous treatment. Data represent means±SD(n=4). Cell viability was greater than 95%.

FIG. 18 shows the time-dependent changes in cell proliferation and Hbproduction. NaPA (5 mM) was added on days 2, 4, 6, and 8 of phase IIcultures derived from normal donors1, and the cells were analyzed on day13. Panel A: Number of Hb-containing cells per ml (×10⁻⁴), and theamounts of Hb (pg) per cell (MCH). Panel B; Total Hb (pg) per mlculture, and the proportion of HbF out of total Hb (%HbF). Data pointsrepresent the means of four determinations. The deviation of results ofeach determination from the mean did not exceed 10%. NaPB at 2.5 mMproduced comparable effects (not shown) In all cases, cell viability wasover 9%.

FIG. 19 shows the effect of NaPA on the proportions of Hb species incultured erythroid precursors derived from a patient with sickle cellanemia. NaPA was added to 7 day phase II cultures. The cells wereharvested and were determined following separation on cation exchangeHPLC.

FIG. 20 shows the increased production of TGF-α by human keratinocytesupon treatment with NaPA and NaPB. Epithetial HK5 cells were treatedwith NaPB (3.0 mM, 1.5 nM, 0.75 mM), NaPB (10 mM, 5.0 mM, 2.5 mM) andPAG (5 nM) continuously for 4 days. Untreated cells served as a control.The amount of TGF-α (ng/ml/10⁶ cells) was measured by using anti-TGF-αantibodies.

FIG. 21 shows the enhanced expression of the surface antigens W6/32 (MHCclass I), DR (MHC class II) and ICAM-1 in melanoma cells treated withNaPB. Melanoma 1011 cells were treated with 2 mM PB for 10 days.Treatment was discontinued for 3 days to document the stability of theeffect. FACS analysis revealed markedly increased expression of theantigens following treatment (shaded area); the expression of thesurface antigens was similar or slightly greater on day 13 than on day10, indicating that PB induced terminal differentiation.

FIG. 22 shows the activation of the Peroxisomal Proliferator Receptor(PPAR) by PA, PB and various phenylacetic acid analogs The activation ismeasured by the increased production of the indicator gene forcloramphenicol acetyl transferase (CAT), which is controlled by theresponse element for acyl-CoA oxidase, relative to the control (C). Theexperimental details for this activation measurement method can be foundin Sher et al., Biochem., 32(21):5598 (1993)). The concentration (in mM)of a particular drug is noted next to the following symbols for thevarious drugs: CF-clofibrate, PA=phenylacetate, CP=chlorophenylacetate,PR=phenylbutyrate, CPB=chlorophenylbutyrate, PAG=phenylacetylglutamine,IPB=iodophenylbutyrate, B=butyrate, IAA=indole acetic acid,NA=naphthylacetate, PP=phenoxypropionic acid, 2-4D=2,4-dichlorophenoxyacetate.

FIG. 23 shows the modulation by phenylbutyrate of glutathione (GSH),gamma-glutamyl transpeptidase (GGT) and catalase activites. Theantioxidant capacity (mM or units/mg protein) of the enzymes weremeasured for up to approximately 100 hours following treatment ofprostatic PC3 cells with 2 mM NaPB.

FIG. 24 shows the radiosensitization by PA and PB of human glioblastomaU87 cells by pretreatment for 72 hours with 1, 3 and 5 mM PA and 2.5 mMPB prior to exposure to ionizing radiation (Co.sup.α γradiation).

FIG. 25 shows the inhibition of the growth of breast MCF-7adriamycin-resistant cancer cells by continuous exposure of up to 10 mMPA for 4 days.

FIG. 26 shows the relationship between lipophilicity and the cytostasisinduced by phenylacetate derivatives in prostate carcinoma cells and inplants. The log 1/IC₅₀ values for prostatic cells (calculated from datapresented in Table 21), were compared with the 1/IC₄ for rapidlydeveloping plant tissues Tested compounds, listed in an increasing orderof their CLOGPs, included 4-hydroxy-PA, PA, 4-fluoro-PA, 3-methyl-PA,4-methyl-PA, 4-chloro-PA, 3-chloro-PA, and 4lodo-Pa.

FIG. 27 shows the phenotypic reversion induced by phenylacetate andselected derivatives. The malignant prostatic PC3 cells were treated asdescribed in "Material and Methods". Data indicates the relative potencyof tested compounds in significantly inhibiting PC3anchorage-independence (A) and completely blocking matrigel invasion(B). Phenylacetate and analogs are presented in an increasing order ofCLOGP (top to bottom). CLOGP values are provided in Tables 21 and 22.The effect on anchorage dependency was confirmed with U87 cells (notshown).

FIG. 28 shows a dose-response curve of the effect of NAPA on theincorporation of ³ H!thymidine in LA-N-5 cells after 7 days oftreatment. Values represent the mean+S.E.M. of quadruplicate samples ofa typical experiment.

FIG. 29 shows that NaPA and RA are synergistic in inhibiting growth ofLA-N-5 cells. Cells were cultured in the presence of RA (10⁻⁷ M), NaPA(1.25 mM), RA (10⁻⁷ M)+NaPA (1.25 mM), or in the absence of addedcompounds as indicated. After 6 days, cultures were assayed forincorporation of ³ H!thymidine. Columns represent the mean (+S.E.M.) oftriplicate samples.

FIG. 30 shows dose response curves showing the effects of NaPA alone (∘)and in the presence of 10⁻⁷ M (∇) and 10⁻⁶ M () RA on specific AChEactivity in LA-N-5 cells after 7 days of culturing. Each pointrepresents the mean of three replicate cultures (S.E.M.<10% in allcases).

FIG. 31 shows the quantitation of N-myc nuclear staining intensitiesfrom cultures shown in FIG. 50. A total of 500 cells per treatmentcondition as indicated were chosen at random for analysis. Results areexpressed as % of total cells counted versus relative staining intensitywith median relative intensities for each treatment condition asfollows: 44, control; 36, NaPA; 29, RA; 16, RA+NaPA.

FIG. 32 shows dose-response curves showing the effects of PA (solidlines) and phenylbutyrate (PB) (dashed lines) on the incorporation of ³H!thymidine into triangles, SK-N-AS; solid diamonds, LA-N-5; hollowdiamonds, Lan-1-15N, solid squares, LA N-6; hollow squares, SK-N-SH-F;solid circles, SK-N-SH-N; and hollow circles, LA-N-2 cells after 7 daysof treatment. In all cases, PB was a more potent inhibitor of cellproliferation than PA.

FIG. 33 shows the time course of PA and PB induced growth inhibition onLA-N-5 neuroblastoma. Cells were treated with the indicatedconcentrations of phenylbutyrate (A) or phenylacetate (B) on day 0 andassayed for incorporation of ³ H!thymidine on a daily basis as shown.

FIG. 34 shows the time course of specific AChE activity of LA-N-5 cellsin the absence or presence of various concentrations of PB (A) or PA (B)as indicated. In all cases, increased in AChE temporarily precededinduced reduction of ³ H!thymidine incorporation as shown in FIG. 33.The sharp decline in AChE activity seen within 4 mM PB after 4 days oftreatment is probably due to reduced viability of the cultures.

FIG. 35 shows the reversibility of the antiproliferative effects ofphenylacetate and phenylbutyrate on LA-N-5 cells. The cells werecultured in the absence (∘) or presence of PA (solid circles; 5 mM) orPB (solid squares; 2 mM) for 6 days, then washed and refed with eithercontrol medium (solid lines) or medium containing the same concentrationof agent as during the treatment phase (dashed lines). Followingwashing, the cells were assayed for incorporation of ³ H!thymidine afterculturing for various days as indicated up to a posttreatment period of2 weeks.

FIG. 36 shows the reveroibility of induced increases in specific AChEactivity. LA-N-5cell were cultured in the absence (solid square) orpresence of PA (vertical striped square; 5 mM) or PB (crossed stripedsquare; 2 mM) for G days, then washed and refed with either controlmedium (solid bar graph) or medium containing the same concentration ofagent as during the treatment phase (line graph with box symbols). Thecells were assayed for specific AChE activity after culturing forvarious days as indicated up to a posttreatment period of 1 week.

FIG. 37 shows the synergistic effect of treatment of prostatic carcinomaPC3 cells with PA along with apigenin and 9-cis-retinoic acid.

FIG. 30 shows the growth arrest of A172 glioma cells upon treatment withNaPA, LOV and a combination of the two drugs (3 days continuoustreatment)

FIG. 39 shows the suppression of glioma cell invasiveness (A172 cells, 3days continuous treatment) by phenylacetate (2 mM) in combination withlovastatin (0.1 μM).

FIG. 40 shows induced melanogenesis (time and dose dependencies).

FIG. 41 shows the potentiation of other drugs.

FIG. 42 shows the dose-response curves of hydroxyurea in three prostatecancer cell lines (PC-3, PC-3M, and DU-145) compared to the controlwells the IC₅₀ for all three cells is approximately 100 μM.

FIG. 43 shows the duration of drug exposure versus survival curve ofhydroxyurea (100 μM) in PC-3 cells. The cells were exposed to drug forvarying periods of time, but all were grown for a total of 5 days (120hours). Drug exposure was terminated at the various intervals byreplacing drug-containing medium with drug-free medium. This experimentdid not detect any recovery in cell viability following brief periods ofdrug exposure. The IC₅₀ was only achieved after 120 hours of exposure.

FIG. 44A shows a plasma concentration versus time simulation of thehydroxyurea dosing regimen employed by Lener et al. (80 mg/kg everythird day) depicted over: 30 days. FIG. 44B shows a plasma concentrationversus time simulation of the hydroxyurea dosing regiment required toproduce plasma concentrations above 100 μM for 5 days in an average 70kg man (1.0 g loading dose followed by 500 mg every 6 hours for 9 days)depicted over 30 days. FIG. 44C shows a plasma concentration versus timesimulation of the hydroxyurea dosing regimen required to produce plasmaconcentrations above 50 μM for 5 days in an average 70 kg man (400 mgloading dose followed by 200 mg every 6 hours for 5 days) depicted over30 days.

FIG. 45 shows the potentiation of antitumor activity of the combinationof hydroxyurea with phenylbutyrate in PC-3 cells.

FIG. 46 shows the growth inhibitory effects of PA on variousneuroectodermal tumor cell lines. The cells were grown in 96 wellmicrotiter plates in MEM+10% FCS and treated with various concentrationsof PA or media alone for 96 h. Then 1 μCi of ³ H-thymidine was added perwell. After 4 h the cells were harvested using a semiautomated cellharvester and the amount of ³ H-thymidine uptake determined by liquidscintillation counting.

FIG. 47 shows how neutralizing antibodies against human TGFβ1 (upperpanel) and TGFβ2 (lower panel) failed to antagonize theantiproliferative effect of PA on U251 cells. U251 cells were culturedin 96 well microtiter plates in MEM+10% FCS in the presence of 10 mM PAor media alone. Neutralizing antibodies directed against TGFβ1 or TGFβ2were added to the PA containing wells at the beginning of the incubationperiod. Normal IgG of the corresponding species served as a control. Therate of cell proliferation was determined by adding 1 uCi of3H-thymidine per well and harvesting the cells after 4 h 3H-thymidineincorporation was determined by liquid scintillation counting.

V. DETAILED DESCRIPTION OF THE INVENTION

As used herein, the term "phenylacetic acid derivative" (or"phenylacetic acid analog") refers to a compound of the formula:##STR3## wherein R₀ is aryl (e.g., phenyl, napthyl), phenoxy,substituted aryl (e.g., one or more halogen e.g., F, Cl, Br, I!, loweralkyl e.g., methyl, ethyl, propyl, butyl! or hydroxy substituents) orsubstituted phenoxy (e.g., one or more halogen e.g., F, Cl, Br, I!,lower alkyl e.g., methyl, ethyl, propyl, butyl! or hydroxysubstituents);

R₁ and R₂ are each H, lower alkoxy (e.g., methoxy, ethoxy), lowerstraight and branched chain alkyl (e.g., methyl, ethyl, propyl, butyl)or halogen (e.g., F, Cl, Br, I);

R₃ and R₄ are each H, lower straight and branched chain alkyl (e.g.,methyl, ethyl, propyl, butyl), lower alkoxy (e.g., methoxy, ethoxy) orhalogen (e.g., F, Cl, Br, I); and

n is an integer from 0 to 2; salts thereof (e.g., Na⁺, K⁺ or otherpharmaceutically acceptable salts); stereoisomers thereof; and mixturesthereof.

When n is equal to 2, each of the two R₃ substituents and each of thetwo R₄ substituents can vary independently within the above phenylaceticacid derivative definition. It is intended that this definition includesphenylacetic acid (PAA) and phenylbutyric acid (PBA). Mixtures accordingto this definition are intended to include mixtures of carboxylic acidsalts, for instance, a mixture of sodium phenylacetate and potassiumphenylacetate. Because the carboxylic portion of these compounds is theprimarily active portion, references herein to a carboxylate, such asphenylacetate (PA) or phenylbutyrate (PB), are intended to refer also toan appropriate counter cation, such as Na⁺, K⁺ or anotherpharmaceutically acceptable cation such as an organic cation (e.g.,arginine). Thus, as used herein, a PA or PB derivative or analog refersto the phenylacetic acid derivatives of this definition. Some of thesederivatives can be interconverted when present in a biological system.For instance, PA can be enzymatically converted to PB within an animaland, similarly, PB can be converted to PA.

Thus, phenylacetic acid derivatives include, without limitation,phenylacetic acid, phenylpropionic acid, phenylbutyric acid,1-naphthylacetic acid, phenoxyacetic acid, phenoxypropionic acid,phenoxybutyric acid, 4-chlorophenylacetic acid, 4-chlorophenylbutyricacid, 4-iodophenylacetic acid, 4-iodophenylbutyric acid,α-methylphenylacetic acid, α-methoxyphenylacetic acid,α-ethylphenylacetic acid, α-hydroxyphenylacetic acid,4-fluorophenylacetic acid, 4-fluorophenylbutyric acid,2-methylphenylacetic acid, 3-methylphenylacetic acid,4-methylphenylacetic acid, 3-chlorophenylacetic acid,3-chlorophenylbutyric acid, 2-chlorophenylacetic acid,2-chlorophenylbutyric acid and 2,6-dichlorophenylacetic acid, and thesodium salts of the these compounds.

The compounds of the present invention can be administeredintravenously, enterally, parenterally, intramuscularly, intranasally,subcutaneously, topically, intravesically or orally. The dosage amountsare based on the effective inhibitory concentrations observed in vitroand in vivo in antitumorigenicity studies. The varied and efficaciousutility of the compounds of the present invention is further illustratedby the findings that they may also be administered concomitantly or incombination with other antitumor agents (such as hydroxyurea,5-azacytidine, 5-aza-2'-deoxycytidine, and suramin); retinoids;hormones; biological response modifiers (such as interferon andhematopoietic growth factors); and conventional chemo- and radiationtherapy or various combinations thereof.

The present invention also provides methods of inducing tumor celldifferentiation in a host comprising administering to the host atherapeutically effective amount of PAA or a pharmaceutically acceptablederivative thereof.

The present invention also provides methods of preventing the formationof malignancies by administering to a host a prophylactically effectiveamount of PAA or a pharmaceutically acceptable derivative thereof.

The present invention also provides methods of treating malignantconditions, such as prostatic cancer, melanoma, adult and pediatrictumors, e.g., brain tumors of glial origin, astrocytoma, Kaposi'ssarcoma, lung adenocarcinoma and leukemias, as well as hyperplasticlesions, e.g., benign hyperplastic prostate and papillomas byadministering a therapeutically effective amount of PAA or apharmaceutically acceptable derivative thereof.

In addition, the present invention provides methods of treatingconditions such as neuroblastoma, promyelocytic leukemia,myelodisplasia, glioma, prostate cancer, breast cancer, melanoma, andnon-small cell lung cancer.

It is understood that the methods and compositions of this invention canbe used to treat animal subjects, including human subjects.

According to the present invention, phenylacetic acid derivatives, andin particular NaPA and NaPB, have been found to be excellent inhibitorsof the growth of specific tumor cells, affecting the proliferation ofthe malignant cells while sparing normal tissues Also, according to thepresent invention, NaPA and its analogs have been found to induce tumorcell differentiation, thus offering a very desirable approach to cancerprevention and therapy. Additionally, NaPA and its analogs have beenfound to be of value for the treatment of viral indications such asAIDS. NaPA is also implicated in the treatment of severe beta-chainhemoglobinopathies. The exact mechanisms by which the compounds used inthe methods of this invention exert their effects are uncertain. Onepotential mechanism may involve depletion of plasma glutamine. Based onthe data reported herein, it is believed that glutamine depletion alonecannot explain the molecular and phenotypic changes observed in vitrofollowing exposure to NaPA. It will be understood, however, that thepresent invention is not to be limited by any theoretical basis for theobserved results.

In specific embodiments, the present invention emcompasses the followingsubject matter:

The present invention provides a method of treating a neoplasticcondition in a subject comprising administering a therapeutic amount ofa phenylacetic acid derivative of the formula:

General Structure A: ##STR4## wherein R₀ =aryl, phenoxy, substitutedaryl or substituted phenoxy;

R₁ and R₂ =H, lower alkoxy, lower straight and branched chain alkyl orhalogen;

R₃ and R₄ =H, lower alkoxy, lower straight and branched chain alkyl orhalogen; and

n=an integer from 0 to 2; salts thereof; stereoisomers thereof; andmixtures thereof. This general structure is hereinbelow referred to asGeneral Structure A without reference to any particular method orcomposition. The neoplastic conditions treatable by this method includeneuroblastoma, acute promyelocytic leukemia, acute myelodisplasia, acuteglioma, prostate cancer, breast cancer, melanoma, non-small cell lungcancer, medulloblastoma, and Burkitt's lymphoma. The compounds for theabove method (as disclosed in General Structure A), and for any of themethods and compositions disclosed herein, specifically include sodiumphenylacetate and sodium phenylbutyrate.

As used throughout this application, the phrase "in combination with"refers to treatment with the constituent drugs of the combination eithersimultaneously or at such intervals that the drugs are simultaneouslyactive in the body. Furthermore, as used herein, the term "therapeuticamount" refers to an amount of an agent, drug or other compound of thegeneric class which is suitable for the claimed use. Therefore,"therapeutic amount" excludes members of the class which do not have therecited activity.

Further provided is a method of preventing a neoplastic condition in asubject comprising administering a prophylactic amount of a phenylaceticacid derivative of General Structure A. This method encompasses a methodwhere the compound is administered in combination with ananti-neoplastic agent.

The present invention also provides a method of inducing thedifferentiation of a cell comprising administering to the cell adifferentiation inducing amount of a phenylacetic acid derivative ofGeneral Structure A.

Also included is a method of inducing the production of fetal hemoglobinin a subject comprising administering to the subject a fetal hemoglobininducing amount of a phenylacetic acid derivative of General StructureA.

The present invention includes a method of treating a pathologyassociated with abnormal hemoglobin activity in a subject comprisingadministering to the subject a therapeutic amount of a phenylacetic acidderivative of General Structure A. This method may be used to treat apathology which is anemia. More specifically, the anemia may be selectedfrom the group consisting of sickle cell and beta thalassemia.

Further provided is a method of treating a viral infection in a subjectcomprising administering to the subject a therapeutic amount of aphenylacetic acid derivative of General Structure A. The viral infectiontreated by the above method may be an infection by a retrovirus.Specifically, this method may be used to treat a subject infected by aHuman Immunodeficiency Virus.

The present invention provides a method of preventing a viral infectionin a subject comprising administering to the subject a prophylacticamount of a phenylacetic acid derivative of General Structure A.

In another embodiment, the present invention provides a method ofinhibiting the production of IL-6 in a cell comprising contacting thecell with an IL-6 inhibiting amount of a phenylacetic acid derivative ofGeneral Structure A. This method of inhibiting may be used in a subjecthaving any of the following pathologies: rheumatoid arthritis,Castleman's disease, mesangial proliferation, glomerulonephritis,uveitis, sepsis, automimmunity inflammatory bowel, type I diabetes,vasculitis and a cell differentiation associated skin disorder. Theinhibition, of course, will be sufficient to treat the disorder.

The present invention also provides a method of inducing the productionof TGFα in a cell comprising contacting the cell with a TGFα inducingamount of a phenylacetic acid derivative of General Structure A. Thismethod may be used where the induction is in a wound of a subject andthe induction is sufficient to promote wound healing.

The present invention also provides a method of inhibiting theproduction of TFG-β2 in a cell comprising contacting the cell with aTGF-β2 inhibiting amount of a phenylacetic acid derivative of GeneralStructure A.

Further provided is a method of treating an AIDS-associated dysfunctionof the central nervous system in a subject comprising administering tothe subject a therapeutic amount of a phenylacetic acid derivative ofGeneral Structure A.

Another embodiment of the present invention is a method of enhancingimmunosurveillance in a subject comprising administering to the subjectan immunosurveillance enhancing amount of a phenylacetic acid derivativeof General Structure A.

The present invention also provides a method of monitoring thebioavailability of a compound of General Structure A. This method isapplicable for the treatment of a pathology not associated withhemoglobin and it comprises administering to a subject the compound andmeasuring the level of fetal hemoglobin, an increase in the amount offetal hemoglobin indicating an increased bioavailability of the compoundto treat the pathology and a decrease in the amount of fetal hemoglobinindicating a decrease in the bioavailability of the compound to treatthe pathology. This method is useful for monitoring a pathology which isa neoplastic condition.

The present invention provides a method of promoting the healing of awound in a subject comprising administering to a wound in the subject awould healing amount of a phenylacetic acid derivative of GeneralStructure A.

Further provided is a method of treating a neoplastic condition in asubject resistant to radiation and chemotherapy comprising administeringto said subject a therapeutic amount of a phenylacetic acid derivativeof General Structure A. This method is particularly useful for treatmentof a neoplastic condition exhibiting the multiple drug resistantphenotype.

As used herein, the terms "retinoid" or "retinoids" includes anysuitable members of the generic class including, but not limited to:9-cis-retinoic acid and all-trans-retinoic acid. Retinoid combinationtherapy is suitable for the treatment of cancers including breastcancer, leukemia and malignant melanoma.

As used herein, inhibitors of the mevalonate pathway include compoundssuch as terpenes and vastatins. Suitable vastatins include lovastatin.Suitable terpenes include limonene and its analogs.

The bioflavonoids are a class of compounds also known as the vitamin Pcomplex and are known for their influence on capillary fragility(hemostatic). They are generally known to decrease capillarypermeability and fragility. As used herein, flavonoids include apigeninand quercetin. However, other flavonoids would be expected to elicit thesimilar utility.

The particular activity of each of the compounds can be screened usingthe assays and models described in the Examples.

As used herein, "modulating lipid metabolism" describes the ability of atherapy to alter lipid production and degradation in vivo. For example,one modulation of lipid metabolism is the reduction of serumtriglycerides (the level of low and high density lipoproteins in asubject's serum), i.e. the lowering of a subject's cholesterol level.

Thus, the present invention also includes the following embodiments:

A method of treating a neoplastic condition in a subject comprisingadministering a therapeutic amount of a retinoid in combination with atherapeutic amount of a phenylacetic acid derivative of GeneralStructure A. The retinoid may be all-trans-retinoic acid or9-cis-retinoic acid. Furthermore, this method may be used to treatneoplastic conditions such as neuroblastoma.

The present invention also provides a method of treating a neoplasticcondition in a subject comprising administering a therapeutic amount ofan inhibitor of the mevalonate pathway in combination with a therapeuticamount of a phenylacetic acid derivative of General Structure A. Thismethod may be practised using inhibitors which are vastatins or ananalogs thereof. One particularly suitable vastatin useful for thismethod is lovastatin. Another class of inhibitors is the terpenes, and,in particular, limonene. Other inhibitors can be screened using themethods set forth in the examples. This method may be used to treatneoplastic conditions including malignant glioma, adenocarcinoma andmelanoma. In addition, the neoplastic condition may be of anon-malignant nature, including, but not limited to, such conditions asnon-malignant glioma, benign prostatic hyperplasia, and papillomavirusinfection. A related method uses the above steps and further includesthe steps of continuously monitoring the subject forrhabdomyolysis-induced myopathy and in the presence ofrhabdomyolysis-induced myopathy, administering ubiquinone to thesubject.

A further method of the present invention is a method of inhibitingHMG-coA reductase and MVA-PP decarboxylase in a subject with aneoplastic condition, comprising administering a therapeutic amount ofan inhibitor of the mevalonate pathway in combination with a therapeuticamount of a phenylacetic acid derivative of General Structure A.Suitable inhibitors-include the class of vastatins and their analogs,and, in particular lovastatin. Other suitable inhibitors are theterpenes and their analogs, and, in particular, limonene. This methodmay be used to treat neoplastic conditions, if necessary, includingmalignant glioma, adenocarcinoma and melanoma. A related method alsoincludes the additional steps of continuously monitoring the subject forrhabdomyolysis-induced myopathy and in the presence ofrhabdomyolysis-induced myopathy, administering ubiquinone to thesubject.

The present invention also provides a method of treating a neoplasticcondition in a subject, comprising administering a therapeutic amount ofa flavonoid in combination with a therapeutic amount of a phenylaceticacid derivative of General Structure A. Suitable flavonoids includeapigenin and quercetin. This method may be used to treat neoplasticconditions including prostatic carcinoma.

The present invention provides a further method of treating a neoplasticcondition in a subject, comprising administering a therapeutic amount ofhydroxyurea in combination with a therapeutic amount of a phenylaceticacid derivative of General Structure A. This combination therapy methodmay be used to treat neoplastic conditions including prostaticcarcinoma.

In another embodiment, the present invention provides a method ofmodulating lipid metabolism in a subject, comprising administering atherapeutic amount of a phenylacetic acid derivative of GeneralStructure A. In a related embodiment, the present invention provides amethod of reducing serum triglycerides in a subject, comprisingadministering a therapeutic amount of a phenylacetic acid derivative ofGeneral Structure A.

The present invention provides a method of locally treating a neoplasticcondition of an internal tissue of a subject, comprising administering,intravesically, a therapeutic amount of a phenylacetic acid derivativeof General Structure A. This method may be used to locally treatneoplastic conditions of externally accessible internal orifices andbladders. Thus, the intravesicle method may be used to treat neoplasticconditions such as bladder carcinoma and kidney cancer.

The present invention provides a method of sensitizing a subject toradiation therapy, comprising administering a therapeutic amount of aphenylacetic acid derivative of General Structure A.

Also provided is a product for simultaneous, separate, or sequential usein treating a neoplastic condition in a subject, comprising, in separatepreparations, a therapeutic amount of a vastatin and a therapeuticamount of a phenylacetic acid derivative of General Structure A. To thiscomposition may be added a therapeutic amount of ubiquinone. Atherapeutic amount of ubiquinone is an amount sufficient to permittolerance of increased dosage of vastatin (or, specifically, lovastatin)without substantial, concomitant side effects.

A further product for simultaneous, separate, or sequential use intreating a neoplastic condition in a subject, comprises, in separatepreparations, a therapeutic amount of a retinoid and a therapeuticamount of a phenylacetic acid derivative of General Structure A. Thiscomposition can be made with retinoids including all-trans-retinoic acidand 9-cis-retinoic acid (or both).

Another novel product for simultaneous, separate, or sequential use intreating a neoplastic condition in a subject is provided. This productcomprises, in separate preparations, a therapeutic amount of hydroxyureaand a therapeutic amount of a phenylacetic acid derivative of GeneralStructure A.

The present invention provides a composition, comprising a therapeuticamount of a vastatin and a therapeutic amount of a phenylacetic acidderivative of General Structure A.

The present invention further provides a composition, comprising atherapeutic amount of a retinoid and a therapeutic amount of aphenylacetic acid derivative of General Structure A.

Finally, the present invention provides a composition, comprising atherapeutic amount of hydroxyurea and a therapeutic amount of aphenylacetic acid derivative of General Structure A.

VI. Examples

The herein offered examples, including experiments, provide methods forillustrating, without any implied limitation, the practice of thisinvention focusing on phenylacetic acid and its derivatives directed toA. Cancer therapy and prevention; B. Treatment and prevention of AIDS;C. Induction of fetal hemoglobin synthesis in β-chainhemoglobinopathies; D. Use of phenylacetic acid and its derivatives inwound healing; E. Use of phenylacetic acid and its derivatives intreatment of diseases associated with interleukin-6; F. Use ofphenylacetic acid and its derivatives in the treatment ofAIDS-associated CNS dysfunction; G. Use of phenylacetic acid and itsderivatives to enhance immunosurveillance; H. Method of monitoring thedosage level of phenylacetic acid and its derivatives in a patientand/or the patient's response to these drugs; I. The activation of thePPAR by phenylacetic acid and its derivatives; J. Use of phenylaceticacid and its derivatives in treatment of cancers having a multiple-drugresistant phenotype; K. phenylacetic acid and its derivatives,correlation between potency and lipophilicity, L. phenylacetic acid andits derivatives in synergistic combination with lovastatin for thetreatment and prevention of cancers such as malignant gliomas or otherCNS tumors, M. phenylacetic acid and its derivatives in synergisticcombination with retinoic acid for the treatment and prevention ofcancers such as those involving neuroblastoma cells, N. phenylaceticacid and its derivatives for the treatment and prevention of cancers andother differentiation disorders such as those involving malignantmelanoma or other neuroectodermal tumors, O. phenylacetic acid and itsderivatives in synergistic combination with hydroxyurea (HU) for thetreatment and prevention of cancers such as prostate cancer, P.phenylacetic acid and its derivatives for the treatment and preventionof cancers involving medulloblastoma and astrocytoma derived cells, Q.phenylacetic acid and its derivatives in human studies relating totreatments with PA and PB, R. phenylacetic acid and its derivatives inmethods of altering lipid metabolism, including reducing serumtriglycerides, and S. methods of administering phenylacetic acid and itsderivatives.

Section A: Phenylacetate in Cancer Prevention and Maintenance Therapy

Recent advances in molecular techniques enable the detection of geneticdisorders associated with a predisposition to cancer. Consequently, itis now possible to identify high-risk individuals as well as patients ina state of remission but afflicted with a residual disease. Despite suchremarkable capabilities, there is still no acceptable preventivetreatment. Chemopreventive drugs are also needed for adjuvant therapy,to minimize the carcinogenic effects of the prevailing anticancer agentsand yet maintain tumor responses.

To qualify for use in chemoprevention, a potential drug should haveantitumor activities, be non-toxic and well tolerated by humans, easy toadminister (e.g., orally or intravenously), and inexpensive. We suggestthat NaPA possesses all of the above characteristics.

1. Prevention of Neoplastic Transformation--Oncogene Transfer Studies

NIH 3T3 cells carrying activated Ejras oncogene (originally isolatedfrom human bladder carcinoma) were used as a model to study thepotential benefit of NaPA treatment to high risk individuals, in whompredisposition is associated with oncogene activation. Cell treatmentwith NaPA was initiated 24-48 hours after oncogene transfer. Results,scored 14-21 days later, show dose-dependent reduction in the formationof ras-transformed foci in cultures treated with NaPA. Molecularanalyses indicated that the drug did not interfere with oncogene uptakeand transcription, but rather prevented the process of neoplastictransformation. The effect was reversible upon cessation of treatment.In treated humans, however, the fate of the premalignant cells may besubstantially different due to involvement of humoral and cellularimmunity (see discussion below).

2. Prevention of tumor progression by genotoxic chemotherapy

Current approaches to combat cancer rely primarily on the use ofchemicals and radiation, which are themselves carcinogenic and maypromote recurrences and the development of metastatic disease. Oneexample is the chemotherapeutic drug 5-aza-2'-deoxycytidine (5AzadC).While this drug shows promise in treatment of some leukemias and severeinborn anemias, the clinical applications have been hindered by concernsregarding toxicity and carcinogenic effects. However, for the first timethe data indicate that NaPA can prevent tumor progression induced bytreatment with 5AzadC.

The experimental model involved nonmalignant 4C8a10 cells (revertants ofHa-ras-transformed NIH 3T3 fibroblasts). Transient treatment of thepremalignant cells with 5AzadC resulted in malignant conversion evidentwithin 2 days, as determined by cell morphology, loss of contactinhibition and anchorage dependent growth in culture, and acquiredinvasive properties and tumorigenicity in recipient athymic mice.Remarkably, NaPA prevented the development of these malignant phenotypesin the 5AzadC treated cultures (Table 1).

                  TABLE 1    ______________________________________              Tumor Formation.sup.a                             Growth    Treatment   Incidence  Size (mm) on matrigel.sup.b    ______________________________________    None        3/8        1 (0.5-2) -    5AzadC (0.1 uM)                8/8        11.5 (4-19)                                     +    NaPA (1.5 mg/ml)                0/8                  -    5AzadC + NaPA                0/8        0         -    (0.1 uM) (1.5 mg/ml)    ______________________________________     .sup.a Cells pretreated in culture were injected s.c. (5 × 10.sup.5     cells per site) into 3 month old female athymic nude mice (Division of     Cancer Treatment, NCI Animal Program, Frederick Cancer Research Facility)     Results indicate the incidence (tumor bearing/injected animals), as well     as tumor size as mean (range), determined after 3 weeks.     .sup.b Cells were plated on top of matrigel (reconstituted basement     membrane) and observed for malignant growth pattern, i.e., active     replication, development of characteristic processes, and invasion.

3. Activity in Humans

In terms of cancer prevention, the beneficial effect of NaPA to humansmay be even more dramatic than that observed with the experimentalmodels. In humans, NaPA is known to deplete circulating glutamine, anamino acid critical for the development and progression of cancer. Theenzymatic reaction leading to glutamine depletion takes place in theliver and kidney. It is not clear whether or not glutamine depletionoccurs in the cultured tumor cells. Moreover, molecular analysisrevealed that NaPA induced the expression of histocompatibility class Iantigens, which are localized on the surface of tumor cells and affectthe immune responses of the host. While the therapeutic benefit of NaPAobserved in cultures is in some cases reversible upon cessation oftreatment, in patients the residual tumor cells would eventually beeliminated by the immune system. Even if chemoprevention will requirecontinuous treatment with NaPA, such treatment would be acceptableconsidering the lack of toxicity.

Pharmaceutical compositions containing phenylacetate have been shown tocause reversal of malignancy and to induce differentiation of tumorcells. To demonstrate the capacity of drugs to induce differentiation oftumor cells, three in vitro differentiation model systems and one invivo phase I clinical trial were used (further described herein). Thefirst system used a human promyelocytic leukemia cell line HL-60. Thiscell line represents uncommitted precursor cells that can be induced toterminally differentiate along the myeloid or monocytic lineage. In thesecond system, immortalized embryonic mesenchymal C3H 10T1/2 cells wereused which have the capability of differentiating into myocytes,adipocytes, or chondrocytes. In the third system, human erythroleukemiaK562 cells were used because they can be induced to produce hemoglobin.Finally, the in vivo experiments demonstrated the efficacy of NaPA ininducing terminal differentiation in humans and animals.

NaPA and NaPB have also been shown to affect tumor growth in vitro andin animal models at pharmacological, non-toxic concentrations. Thesearomatic fatty acids induced cytostasis and promoted maturation ofvarious human malignant cells, including hormone-refractory prostaticcarcinoma, glioblastoma, malignant melanoma, and lung carcinoma. Themarked changes in tumor biology were associated with alterations in theexpression of genes implicated in tumor growth, invasion, angiogenesis,and immunogenicity. Multiple mechanisms of drug action appear to beinvolved. These mechanisms include (a) modification of lipid metabolism,(b) regulation of gene expression through DNA hypomethylation andtranscriptional activation, and (c) inhibition of proteinisoprenylation. Phase I clinical trials confirmed the efficacy of thesenovel, nontoxic differentiation inducers (see Example 15).

Example 1

HL-60 and 10T1/2 cells--PAG and NaPA treatment

Referring now to the data obtained using the first system (resultsillustrated in FIG. 1), logarithmically growing HL-60 --! and 10T1/2-∘-! cells were treated for four days with NaPA solid line! orphenylacetylglutamate (PAG) dashed line!. The adherent cells weredetached with trypsin/EDTA and the cell number determined using ahemocytometer. Data points indicate the mean±S.D. of duplicates from twoindependent experiments. The cell lines were obtained from the AmericanType Culture Collection and maintained in RPMI 1640 (HL-60) orDulbecco's Modified Eagle's Medium (10T1/2) supplemented with 10% heatinactivated fetal calf serum (Gibco Laboratories), 2 mM L-Glutamine, andantibiotics. PAA (Sigma, St. Louis Mo.) and PAG were each dissolved indistilled water, brought to pH 7.0 by the addition of NaOH, and storedin -20° C. until used. As demonstrated in FIG. 1, NaPA treatment of theHL-60 and 10T1/2 cultures was associated with dose dependent inhibitionof cell proliferation.

Example 2

HL-60 cells--induction of granulocyte differentiation

To further evaluate the effectiveness of NaPA as an inducer of tumorcell differentiation, the ability of NaPA to induce granulocytedifferentiation in HL-60 was investigated. The ability of cells toreduce nitroblue tetrazolium (NBT) is indicative of oxidase activitycharacteristic of the more mature forms of human bone marrowgranulocytes. NBT reduction thus serves as an indicator of granulocytedifferentiation. In FIG. 2, the number of NBT positive cells wasdetermined after 4 days solid bars! or 7 days hatched bar! of treatment.NaPA (h), 1.6 mg/ml; NaPA (1), 0.8 mg/ml. 4-hydroxyphenylacetate (4HPA)and PAG were used at 1.6 mg/ml. Potentiation by retinoic acid (RA) 10 nMwas comparable to that by interferon gamma 300 IU/ml. The direction ofdifferentiation towards granulocytes in cultures treated with NaPA,whether used alone or in combination with RA, was confirmed bymicroscopic evaluation of cells stained with Wright Stain and the lackof nonspecific esterase activity. The effect of acivicin (ACV) 1 μg/mlwas similar to 6-diazo-5-oxo-L-norleucine (DON) 25 μg/ml. Glutaminestarvation (Gln,<0.06 mM) was as described. Cell viability determined bytrypan blue exclusion was over 95% in all cases, except for DON and ACVwhich were 75% and 63%, respectively. DON, ACV and HPA are glutamineantagonists. As illustrated in FIG. 2, it is clear that NaPA is capableof inducing granulocyte differentiation in HL-60. As further illustratedin FIG. 2, differentiation of HL-60, assessed morphologically andfunctionally, was sequential and could be further enhanced by theaddition of low doses of retinoic acid RA, 10 nM) or interferon gamma(300 IU/ml). After seven days of NaPA treatment, or four days, whencombined with RA, the HL-60 cultures were composed of early stagemyelocytes and metamyelocytes (30-50%), as well as banded and segmentedneutrophils (30-40%) capable of NBT.

Pharmacokinetics studies in children with urea cycle disorders indicatethat infusion of NaPA 300-500 mg/kg/day, a well tolerated treatment,results in plasma levels of approximately 800 μg/ml. Brusilow, S. W. etal. Treatment of episodic hyperammonemia in children with inborn errorsof urea synthesis. The New England Journal of Medicine. 310:1630-1634(1984).! This same concentration was shown to effectively induce tumorcell differentiation in the present experimental system.

Example 3

10T1/2 cells--NaPA induction of adipocyte conversion

FIG. 3 illustrates that NaPA is capable of inducing adipocyte conversionin 10T1/2 cultures. Confluent cultures were treated with NaPA for sevendays. FIG. 3 shows quantitation of adipocytosis. Cells were fixed with37% formaldehyde and stained with Oil-Red O. The stained intracellularlipid was extracted with butanol, and the optical density was determinedusing a Titertek Multiskan MC, manufactured by Flow Laboratories, at awavelength 510 nm. Increased lipid accumulation was evident in-cellstreated with as little as 0.024 mg/ml of NaPA. The results in FIG. 3show that differentiation was dose- and time-dependent, and apparentlyirreversible upon cessation of treatment. NaPA at 800 μg/ml wasefficient and totally free of cytotoxic effect. In the 10T1/2 model,adipoocyte conversion involved over 80% of the cell population. It wasnoted that higher drug concentrations further increased the efficiencyof differentiation as well as the size of lipid droplets in each cell.

It is known that glutamine conjugation by NaPA is limited to humans andhigher primates and that in rodents NaPA instead binds glycine. (James,M. O. et al. The conjugation of phenylacetic acid in man, sub-humanprimates and some non-primate species. Proc. R. Soc. Lond. B. 182:25-35(1972).! Consequently, the effect of NaPA on the mouse 10T1/2 cell linecould not be explained by an effect on glutamine. In agreement, neitherglutamine starvation nor treatment with glutamine antagonists such asDON and ACV resulted in adipocyte conversion.

Example 4

Induction of lipid accumulation and adipocyte differentiation

4. Clinical use of phenylacetate and derivatives

                  TABLE 2    ______________________________________    Phenylacetate and Derivatives: Induction of cellular    differentiation in premalignant 10T1/2 cells    Compounds      Differentiation at 1 mM                                  DC.sub.50 *    (sodium salts) (%)            (mM)    ______________________________________    Phenylacetate  65             0.7    1-naphthylacetate                   >95            <0.1    3-chlorophenylacetate                   80             0.5    4-chlorophenylacetate                   50             1.0    2,6-dichlorophenylacetate                   75             0.5    4-fluorophenylaceatae                   65             0.7    ______________________________________     *DC.sub.50, concentration of compound causing 50% differentiation

As shown in Table 2, phenylacetate and its derivatives efficientlyinduced lipid accumulation and adipocyte differentiation in premalignantcells. These and other results indicate that the tested compounds mightbe of value in:

A. Cancer Prevention. Non-replicating, differentiated tumor cells arenot likely to progress to malignancy.

B. Differentiation therapy of malignant and pathological nonmalignantconditions.

C. Treatment of lipid disorders, in which patients would benefit fromincreased lipid accumulation.

D. Wound healing. This is indicated by the ability of phenylacetate toinduce collagen synthesis in fibroblasts (see Section D herein).

Studies in plants have revealed that NaPA can interact withintracellular regulatory proteins and modulate cellular RNA levels. Inan attempt to explore the possible mechanism of action, Northern blotanalysis of HL-60 and 10T1/2 cells was performed according toconventional methods. Cytoplasmic RNA was extracted, separated andanalyzed (20 μg/lane) from confluent cultures treated for 72 hours withNaPA or PAG (mg/ml); C is the untreated control. The aP2 cDNA probe waslabeled with ³² P!dCTP (New England Nuclear) using a commerciallyavailable random primed DNA labeling kit. Ethidium bromide-stained 28SrRNA indicates the relative amounts of total RNA in each lane.

The results of the Northern blot analysis of HL-60 and 10T1/2 cells,showed marked changes in gene expression shortly after NaPA treatment.Expression of the adipocyte-specific aP2 gene was induced within 24hours in treated 10T1/2 confluent cultures reaching maximal mRNA levelsby 72 hours.

Example 5

HL-60 cells--myc down regulation

In HL-60, cell transformation has been linked to myc amplification andover-expression, and differentiation would typically require downregulation of myc expression. Collins, S. J. The HL-60 promyelocyticleukemia cell line: Proliferation, differentiation, and cellularoncogene expression. Blood. 70:1233-1244 (1987)!. To demonstrate thekinetics of myc inhibition and HLA-A induction, Northern blot analysisof cytoplasmic RNA (20 μg/lane) was carried out on cells treated withNaPA and PAG for specified durations of time and untreated controls (-).The dose-dependency and specificity of the effect of NaPA was observed.Two concentrations of NaPA, 1.6 mg/ml (++) and 0.8 mg/ml (+), and PAG at1.6 mg/ml were investigated. The ³² P-labeled probes used were myc 3rdexon (Oncor) and HLA-A3 Hind III/EcoRI fragment. NaPA caused a rapiddecline in the amounts of myc mRNA. This occurred within 4 hours oftreatment, preceding the phenotypic changes detectable by 48 hours,approximately two cell cycles, after treatment. Similar kinetics of mycinhibition have been reported for other differentiation agents such asdimethyl sulfoxide, sodium butyrate, bromodeoxyuridine, retinoids, and1,25-dihydroxyvitamin D₃. The results observed suggest that downregulation of oncogene expression by NaPA may be responsible in part forthe growth arrest and induction of terminal differentiation. Inaddition, it is evident in FIG. 5 that NaPA treatment of the leukemiccells was associated with time- and dose-dependent accumulation of HLA-AmRNA coding for class I major histocompatibility antigens. This enhancesthe immunogenicity of tumors in vivo.

Example 6

K562 cells--NaPA promotes hemoglobin biosynthesis

Further support for the use of NaPA as a non-toxic inducer of tumor celldifferentiation is found in the ability of NaPA to promote hemoglobinbiosynthesis in erythroleukemia cells. K562 leukemic cells have anonfunctional beta-globin gene and, therefore, do not normally producesignificant amounts hemoglobin. When K562 human erythroleukemia cellswere grown in the presence of NaPA at 0.8 and 1.6 mg/ml concentrations,hemoglobin accumulation, a marker of differentiation, was found toincrease 4 to 9 fold over that of control cells grown in the absence ofNaPA. Hemoglobin accumulation was determined by Benzidine staining ofcells for hemoglobin and direct quantitation of the protein. The resultsof this study are reported in Table 16.

It has been shown that high concentrations of NaPA inhibit DNAmethylation in plants. Vanjusin, B. J. et al. Biochemia 1, 46:47-53(1981)!. Alterations in DNA methylation can promote oncogenesis in theevolution of cells with metastatic capabilities. Rimoldi, D. et al.Cancer Research. 51:1-7 (1991)!. These observations prompted someconcerns regarding potential long-term adverse effects with the use ofNaPA. To determine the potential tumorigenicity of NaPA, a comparativeanalysis was performed using NaPA and the known hypomethylating agent5-aza-2'-deoxycytidine (5AzadC).

Premalignant cells (3-4×10⁵) were plated in 75 cm² dishes and 5AzadC 0.1μM was added to the 20 and 48 hrs after plating. The cells were thensubcultured in the absence of the nucleoside analog for an additionalseven weeks. Cells treated with NaPA at 1.6 mg/ml were subcultured inthe continuous presence of the drug. For the tumorigenicity assay, 4-5week-old female athymic nude mice were inoculated s.c. with 1×10⁶ cellsand observed for tumor growth at the site of injection.

The results set forth in Table 3 show that NaPA, unlike the cytosineanalog, did not cause tumor progression.

                  TABLE 3    ______________________________________    Tumorigenicity of C3H 10T1/2 Cells in Athymic Mice           Tumors             Incidence             (positive/     Diameter  Time    Treatment             injected mice) (mm ± S.D.)                                      (weeks)    ______________________________________    None     0/8            0         13    5AzadC   8/8            5.5 ± 2.5                                       8    NaPA     0/8            0         13    ______________________________________

The transient treatment of actively growing 10T1/2 cells with 5AzadCresulted in the development of foci of neoplastically transformed cellswith a frequency of about 7×10⁻⁴. These foci eventually became capableof tumor formation in athymic mice. By contrast, actively replicating10T1/2 cultures treated for seven weeks with NaPA, 800-1600 μg/ml,differentiated solely into adipocytes, forming neither neoplastic fociin vitro nor tumors in vivo in recipient mice.

Furthermore, experiments have demonstrated that NaPA can preventspontaneous or 5AzadC-induced neoplastic transformation, thusdemonstrating its novel role in cancer prevention. It is known that thetreatment of premalignant 4C8 and 10T1/2 cells with carcinogens such as5AzadC produces malignant conversion of the respective cells. When 4C8Remold: et al., Cancer Research, 51:1-7 (1990)! and 10T1/2 cells wereexposed to 5AzadC, malignant conversion became evident in two days andtwo weeks, respectively. NaPA (0.8-1.6 mg/ml) prevented the appearanceof the malignant phenotype, as determined by cell morphology, contactinhibition and anchorage dependent growth in culture.

Example 7

Growth arrest in malignant gliomas

In addition, Phenylacetate has been implicated in damage to immaturebrain in phenylketonuria. Because of similarities in growth pattern andmetabolism between the developing normal brain and malignant centralnervous system tumors, phenylacetate may be detrimental to some braincancers. Phenylacetate can induce cytostasis and reversal of malignantproperties of cultured human glioblastoma cells, when used atpharmacological concentrations that are well tolerated by children andadults. Interestingly, treated tumor cells exhibited biochemicalalterations similar to those observed in phenylketonuria-likeconditions, including selective decline in de novo cholesterol synthesisfrom mevalonate. Since gliomas, but not mature normal brain cells, arehighly dependent on mevalonate for production of sterols and isoprenoidsvital for cell growth, phenylacetate would be expected to affect tumorgrowth in vivo, while sparing normal tissues. Systemic treatment of ratsbearing intracranial gliomas resulted in significant tumor suppressionwith no apparent toxicity to the host. The experimental data, which areconsistent with clinical evidence for selective activity againstundifferentiated brain, suggest that phenylacetate may offer a safe andeffective novel approach to treatment of malignant gliomas.

Clinical experience, obtained during phenylacetate treatment of childrenwith urea cycle disorders, indicates that millimolar levels can beachieved without significant adverse effects. The lack of neurotoxicityin these patients is, however, in marked contrast to the severe braindamage documented in phenylketonuria (PKU), an inborn error ofphenylalanine metabolism associated with excessive production ofphenylacetate, microcephaly, and mental retardation. Scriver, C. R., andC. L. Clow. 1980. Phenylketonuria: epitome of human biochemicalgenetics. New Engl. J. Med. 303:1394-1400.! The differences in clinicaloutcome can be explained by the fact that, although phenylacetatereadily crosses the blood-brain barrier in both prenatal and postnatallife, neurotoxicity is limited to the immature brain. Compellingevidence for a developmentally restricted window of susceptibility isprovided by the phenomenon of "maternal PKU syndrome": PKU females whoare diagnosed early and maintained on a phenylalanine-restricted diet,develop normally and subsequently tolerate a regular diet. These womenoften give birth to genetically normal, yet mentally retarded infantsdue to the untreated maternal PKU. The elevated levels of circulatingphenylacetate, while sparing the mature tissues of the mother, aredetrimental to the fetal brain. The primary pathological changes in PKUinvolve rapidly developing glial cells and are characterized byalterations in lipid metabolism and myelination with subsequent neuronaldysfunction. The vulnerable fetal glial tissues resemble neoplasticglial cells in numerous molecular and biochemical aspects, includingunique dependence upon mevalonate (MVA) metabolism for synthesis ofsterols and isoprenoids critical to cell replication Kandutsch, A. A.,and S. E. Saucier. 1969. Regulation of sterol synthesis in developingbrains of normal and jimpy mice. Arch. Biochem. Biophys. 135:201-208;Fumagalli, R., E. Grossi, P. Paoletti, and R. Paoletti. 1964. Studies onlipids in brain tumors. I. Occurrence and significance of sterolprecursors of cholesterol in human brain tumors. J. Neurochem.11:561-565; Grossi, E., P. Paoletti, and R. Paoletti. 1958. An analysisof brain cholesterol and fatty acid biosynthesis. Arch. Int. Physiol.Biochem. 66:564-572!, and on circulating glutamine as the nitrogen donorfor DNA, RNA and protein synthesis Perry, T. L., S. Hasen, B. Tischler,R. Bunting, and S. Diamond. 1970. Glutamine depletion inphenylketonuria, a possible cause of the mental defect. New Engl. J.Med. 282:761-766; Weber, G. 1983. Biochemical strategy of cancer cellsand the design of chemotherapy: G.H.A. Clowes Memorial Lecture. CancerRes. 43:3466-3492!. The hypothesis underlying these studies was thatphenylacetate, known to conjugate and deplete serum glutamine in humans,and to inhibit the MVA pathway in immature brain Castillo, M., M. F.Zafra, and E. Garcia-Peregrin. 1988. Inhibition of brain and liver3-hydroxy-3-methylglutaryl-CoA reductase and mevalonate-5-pyrophosphatedecarboxylase in experimental hyperphenylalaninemia. Neurochem. Res.13:551-555; Castillo, M., J. Iglesias, M. F. Zafra, and E.Garcia-Peregrin. 1991. Inhibition of chick brain cholesterogenic enzymesby phenyl and phenolic derivatives of phenylalanine. Neurochem. Int.18:171-174; Castillo, M., M. Martinez-Cayuela, M. F. Zafra, and E.Garcia-Peregrin. 1991. Effect of phenylalanine derivatives on the mainregulatory enzymes of hepatic cholestrogenesis. Mol Cell. Biochem.105:21-25!, might attack these critical control points in malignantgliomas. The efficacy of phenylacetate was demonstrated using both invitro and in vivo tumor models.

Cell Cultures and Reagents. Human glioblastoma cell lines were purchasedfrom the American Type Culture Collection (ATCC, Rockville, Md.), andmaintained in RPMI 1640 supplemented with 10% heat inactivated fetalcalf serum, antibiotics and 2 mM L-glutamine, unless otherwisespecified. Human umbilical vein endothelial cells, isolated from freshlyobtained cords, were provided by D. Grant and H. Kleinman (NIH, BethesdaMd). Sodium salts of phenylacetic acid and of phenylbutyric acid wereprovided by Elan Pharmaceutical Corporation (Gainseville, Ga.).Phenylacetylglutamine was a gift from S. Brusilow (Johns Hopkins, Md.).

Evaluation of Cell Replication and Viability. Growth rates weredetermined by an enzymatic assay using 3-4,5-dimethylthiazol-2-yl!-2,5-diphenyltertrazolium bromide (Sigma, St.Louis, Mo.) Alley, M. C., D. A. Scudiero, A. Monks, M. L. Hursey, M. J.Czerwinski, D. L. Fine, B. J. Abbott, J. G. Mayo, R. H. Schoemaker, andM. R. Boyd. 1988. Feasibility of drug screening with panels of humantumor cell lines using a microculture tetrazolium assay. Cancer Res.48:589-601!, cell enumeration with a hemocytometer following detachmentwith trypsin/EDTA, and by thymidine incorporation into DNA. Thedifferent assays produced essentially the same results. Cell viabilitywas assessed by trypan blue exclusion.

Colony Formation in Semi-Solid Agar. Tumor cells were detached withtrypsin/EDTA, re-suspended in growth medium containing 0.36% agar, andplaced onto a base layer of solid agar (0.9%) in the presence or absenceof drugs. Colonies composed of 30 or more cells were scored after threeweeks.

Immunocytochemistry. Cells were immunostained with anti-vimentinmonoclonal antibodies using Dako PAP kit K537 (Dako Corporation,Calif.).

Measurement of Cholesterol, Protein and DNA Synthesis. For studies ofsteroid synthesis, cells were labeled for 24 hours with 5×10⁶ DPM 5-³H!-mevalonate (35 Ci/mmol) (New England Nuclear, Boston, Mass.) ingrowth medium containing 3 μM lovastatin and 0.5 mM unlabeledmevalonate, in the presence or absence of 5 mM phenylacetate or 2.5 mMphenylbutyrate. Cellular steroids were extracted with hexane andseparated by silica thin layer chromatography. The R_(f) of thehexane-soluble radiolabled product was identical to that of aradiolabled cholesterol standard in three different solvent systems.Similarly treated cells were tested for de novo protein and DNAsynthesis by metabolic labeling with ³ H!-leucine (158 Ci/mmol) or ³H!-deoxythymidine (6.7 Ci/mmol)(New England Nuclear). Measurements of ¹⁴CO₂ released from 1-¹⁴ C!-mevalonate (49.5 mCi/mmol)(Amersham, Chicago,Ill.) in cell homogenates incubated with phenylacetate/phenylbutyratewere performed with minor modifications to established procedures.

Analysis of Protein Isoprenylation. Cell cultures were incubated with 10mM phenylacetate or 2.5 mM phenylbutyrate for 24 hours in completegrowth medium, and labeled with RS- 2-¹⁴ C!-mevalonate (16 μCi/ml,specific activity 15 μCi/mmol) (American Radiolabeled Chemicals, Inc.St. Louis, Mo.) during the final 15 hours of treatment. Whole cellproteins were extracted, resolved on 10% SDS-polyacrylamide gels, andstained with Commassie Brilliant Blue. Gels were then dried and exposedto Kodak X-Omat film for 4 days.

Animal Studies. To determine the effect of phenylacetate on thetumorigenic phenotype of human glioblastoma cells, cultures werepre-treated for one week and then harvested, resuspended in mediumcontaining 30% matrigel (Collaborative Biomedical Products, Bedford,Mass.), and transplanted s.c. (2.5×10⁶ cells per site) into 5-week oldfemale athymic mice (Division of Cancer Treatment, NCI Animal Program,Frederick Cancer Research Facility). The animals were then observed fortumor growth at the site of injection. To further evaluate drug efficacyin vivo, Fisher 344 rats received a stereotaxic inoculation of syngeneic9 L gliosarcoma cells (4×10⁴) into the deep white matter of the rightcerebral hemisphere, as previously described Weizsaecker, M., D. F.,Deen, M. L. Rosenblum, T. Hoshino, P. H. Gutin, and M. Baker. 1981. The9 L rat brain tumor: description and application of an animal model. J.Neurol. 224:183-192; Culver, K. W., Z. Ram, S. Walbridge, H. Ishii, E.H. Oldfield, and R. M. Blaese. 1992. In vivo gene transfer withretroviral vector producer cells for treatment of experimental braintumors. Science. 256:1550-1552!. The animals were then subjected to twoweeks of continuous treatment with sodium phenylacetate (550 mg/kg/day,s.c.), using osmotic minipumps transplanted subcutaneously. In controlrats the minipumps were filled with saline. Statistical analysis of dataemployed the Fisher's Exact Test.

Induction of cytostasis and phenotypic reversion in cultured humanglioblastoma cells. Treatment of glioblastoma cells with phenylacetateresulted in time-and dose-dependent growth arrest (FIG. 4), accompaniedby similarly diminished DNA synthesis. After 4-6 days of continuoustreatment with 4 mM phenylacetate, there was approximately 50%inhibition of growth in U87, A172, U373, U343, and HS683 cultures (IC₅₀4.4±0.6 mM). Reflecting on the heterogenous nature of tumor cellresponses, glioblastoma U251 and U138 cells were less sensitive withIC₅₀ values of 8-10 mM. Further studies, mimicking pharmacologicalconditions that are expected in patients, involved exposure of cells tophenylacetate in glutamine-depleted medium. These conditions completelyblocked glioblastoma cell growth, but had little effect on thereplication of normal endothelial cells (FIG. 7). Phenylbutyrate, anintermediate metabolite of phenylacetate formed in the brain by fattyacid elongation, also inhibited tumor cell replication (IC₅₀ 2.2 ±0.2 mMin A172, U87 and U373), while the end metabolite, phenylacetylglutamine,was inactive. In addition to inducing selective tumor cytostasis, bothphenylacetate and phenylbutyrate promoted cell maturation and reversionto a nonmalignant phenotype, manifested by an altered pattern ofcytoskeletal intermediate filaments, loss of anchorage-independence, andreduced tumorigenicity in athymic mice (Table 4). Immunocytochemicalanalysis of vimentin in phenylacetate-treated human glioblastoma U87cells showed altered morphology and cytoskeletal filament pattern. Thesechanges, confirmed by immunolabeling for glial fibrillary acidic proteinare consistent with cell maturation and correlate with reducedproliferative capacity and regained contact inhibition of growth. Theseprofound changes in tumor behavior were accompanied by alterations inthe expression of genes implicated in growth control, angiogenesis, andimmunosuppression (e.g., TGFα, HbF, and TGF-β2).

                  TABLE 4    ______________________________________    Reversal of Malignancy of Human Glioblastoma Cells                 Clonogenicity                            Tumor Incidence.sup.2                 in Soft Agar.sup.1                            Positive/Injected    Treatment    (%)        Sites    ______________________________________    None         8.1        9/10    Phenylacetate    2.5 mM       0.5        ND    5 mM         >0.01      2/10    Phenylbutyrate    1.25 mM      0.15       ND    2.5 mM       >0.01      1/10    ______________________________________     .sup.1 U87 cells were detached with trypsin/EDTA, resuspended in growth     medium containing 0.36% agar, and placed onto a base layer of solid agar     (0.9%) in the presence or absence of drugs. Colonies composed of 30 or     more cells were scored after three weeks.     .sup.2 U87 cells pretreated in culture for one week, were harvested,     resuspended in medium containing 30% matrigel, and transplanted s.c. into     5week old female athymic mice (2.5 × 10.sup.6 cells per mouse). Dat     were recorded 5 weeks after cell inoculation.     ND = not determined.

Phenylacetate inhibits the mevalonate pathway and proteinisoprenylation. The most consistent biochemical change observed in glialcells exposed to phenylacetate involved alterations in lipid metabolismand inhibition of the MVA pathway (FIG. 6). Active de novo synthesis ofcholesterol and isoprenoids from precursors such as acetyl-CoA and MVAis an important feature of the developing brain (but not the maturebrain), coinciding with myelination. It is also a hallmark of malignantgliomas Azarnoff, D. L., G. L. Curran, and W. P. Williamson. 1958.Incorporation of acetate-1¹⁴ C into cholesterol by human intracranialtumors in vitro. J. Nat, Cancer Inst. 21:1109-1115; Rudling, M. J., B.Angelin, C. O. Peterson, and V. P. Collins. 1990. Low densitylipoprotein receptor activity in human intracranial tumors and itsrelation to cholesterol requirement. Cancer Res. 50 (suppl):483-487!.Cholesterol production and protein isoprenylation diminished within 24hours of glioblastoma treatment with either phenylacetate orphenylbutyrate (FIG. 7) preceding changes in DNA and total proteinsynthesis, which were detectable after 48 hours. The reduction inisoprenylation was paralleled by a decrease in MVA decarboxylation (toless than 50% of control), an effect previously observed in embryonicbrain in PKU-like conditions. MVA-5-pyrophosphate decarboxylase, a keyenzyme regulating cholesterol synthesis in brain, is inhibited byphenylacetate under conditions in which MVA kinase and MVA-5-phosphatekinase are only minimally affected. Phenylacetate might also interferewith MVA synthesis from acetyl-CoA. Glioblastoma cells could not,however, be rescued by exogenous MVA (0.3-3 mM), suggesting that MVAutilization, rather than its synthesis, is the prime target. The declinein MVA decarboxylation and protein isoprenylation inphenylacetate-treated cells could be mimicked by using 1-2.5 mMphenylbutyrate.

Mevalonate is a precursor of several isopentenyl moieties required forprogression through the cell cycle such as sterols, dolichol, the sidechains of ubiquinone and isopentenyladenine, and prenyl groups thatmodify a small set of critical proteins Goldstein, J. L., and M. S.

Brown. 1990. Regulation of the mevalonate pathway. Nature. 343:425-430;Marshall, C. J. 1993. Protein prenylation: A mediator of protein-proteininteractions. Science. 259:1865-1866; Braun, P. E., D. De Angelis, W. W.Shtybel, and L. Bernier. 1991. Isoprenoid modification permits2',3'-cyclic nucleotide 3'-phosphodiesterase to bind to membranes. J.Neurosci. Res. 30:540-544!. The latter include plasma membrane G andG-like proteins (e.g., ras) involved in mitogenic signal transduction(molecular weight 20-26 kDa), the myelination-related enzyme2',3'-cyclic nucleotide 3'-phosphodiesterase, and nuclear envelopelamins that play a key role in mitosis (44-74 kDa). Inhibition of steroland isoprenoid synthesis during rapid development of the brain couldlead to the microcephaly and impaired myelination seen in untreated PKU.Targeting MVA in dedifferentiated malignant gliomas, on the other hand,would be expected to inhibit tumor growth in vivo without damaging thesurrounding normal tissues, as the MVA pathway is significantly lessactive in mature brain.

Activity of phenylacetate in experimental gliomas in rats. To evaluatethe in vivo antitumor effect of phenylacetate, Fisher rats wereinoculated with stereotaxic intracerebral injection of syngeneic 9 Lgliosarcoma cells. This tumor model is known for its aggressive growthpattern that results in nearly 100% mortality of rats within 3 to 4weeks. Phenylacetate was continuously administered by implantedsubcutaneous osmotic minipumps to deliver a clinically-achievable doseof 550 mg/kg/day. Systemic treatment for two weeks of rats bearingintracranial glioma cells markedly suppressed tumor growth (p<0.05,Table 5 and with no detectable adverse effects. Further studies inexperimental animals indicate that phenylacetate (plasma andcerebrospinal fluid levels of 2-3 mM) induces tumor cell maturation invivo and significantly prolongs survival.

                  TABLE 5    ______________________________________    Phenylacetate Activity in Experimental Brain Cancer                   Brain Tumors.sup.2                No.      Macro-    Micro-                                         Tumor    Treatment.sup.1                of animals                         scopic    scopic                                         Free    ______________________________________    Saline      10       8         1     1    Phenylacetate                15       3         4     8    ______________________________________     .sup.1 Fisher 344 rats received a stereotaxic inoculation of syngeneic 9L     gliosarcoma cells into the deep white matter of the right cerebral     hemisphere, as described in Material and Methods. Animals were then     subjected to two weeks of continuous treatment with either sodium     phenylacetate (550 mg/kg/day, s.c.) or saline, using osmotic minipumps     transplanted subcutaneously.     .sup.2 Animals were sacrificed 23 days after tumor inoculation to     determine antitumor effects. Findings were confirmed by histological     evaluation of the inoculated site.

Summary and Prospective. Phenylacetate has long been implicated indamage to the developing fetal brain. As primary CNS tumors are highlyreminiscent of immature fetal brain, malignant gliomas should be equallyvulnerable. Moreover, viewing maternal PKU syndrome as a natural humanmodel, phenylacetate would be expected to suppress the growth of brainneoplasms without harming normal tissues. Experimental data supportsthis hypothesis. Phenylacetate induced selective cytostasis and promotedmaturation of glioma cells in vitro and in vivo. Premature growth arrestand differentiation could also underlie the damage to fetal brain inPKU. Multiple mechanisms of action are involved, including inhibition ofprotein isoprenylation and depletion of plasma glutamine in humans. Thedemonstrable antitumor activity, lack of toxicity, and ease ofadministration (oral or intravenous), demonstrate the clinical efficacyof phenylacetate in management of malignant gliomas, and perhaps ofother neoplasms as well. Previously, phenylacetate showed activity inprostate cancer in vitro. Phase I clinical studies with phenylacetate inthe treatment of adults with cancer confirmed that therapeutic levelscan be achieved in the plasma and cerebrospinal fluid with nosignificant toxicities, and provide preliminary evidence for benefit toprostatic carcinoma and glioblastoma patients (see Example 18).

Phenylacetate was used to treat human solid tumors, including prostaticcarcinoma, glioblastomas, and malignant melenoma. Treatment resulted inselective cytostasis and phenotypic reversion, as indicated by therestored anchorage-dependence, reduced invasiveness and loss oftumorigenicity in athymic mice. Molecular analysis of brain andhormone-refractory prostate cancer cells revealed marked decline in theproduction and secretion of TGFβ, a protein implicated in growthcontrol, angiogenesis, and immunosuppression. Treated prostatic cellsexhibited decreased proteolytic activity mediated byurokinase-plasminogen activator, a molecular marker of diseaseprogression in man.

Example 8

Growth arrest, tumor maturation, and extended survival in brain tumorstreated with NaPA

In Vitro Studies.

Cell proliferation. The effect of NaPA on cell proliferation wasevaluated using tritiatedthymidine incorporation assay on cultured 9 Lgliosarcoma cells and cell enumeration using a hemocytometer followingdetachment with trypsin/EDTA. 9 L is a syngeneic malignant glial tumorderived from Fischer 344 rats and is associated with 100% mortalitywithin three to four weeks after intracerebral inoculation (WeizsaeckerM., Deen D. F., Rosenblum M. L., et al. The 9 L rat brain tumor:description and application of an animal model. J Neuol. 1981;224:183-1921. Tumor cells were plated at 5×10⁴ tumor cells/well in24-well plates (Costar, Cambridge, Mass.) in Dulbecco Modified Eagle'smedium (DMEM) with 10% fetal bovine serum (Hyclone Laboratories Inc.,Logan, Utah), 2 mM L-glutamine (GIBCO BRL, Gaithersburg, Md.), 50 U/mlpenicillin (GIBCO) and 50 μg/ml streptomycin (GIBCO) and 2.5 μg/mlFungizone (ICN Biomedicals Inc., Costa Mesa, Calif.). After 24 hours,the medium was changed and NaPA (Elan Pharmaceutical Research Corp.,Gainesville, Ga.) added to the medium at 0, 2.5, 5, and 10 mMconcentration for 5 days. Six hours before harvest, 0.5 mCitritiatedthymidine (ICN Radiochemicals, Irvine, Calif.) was added toeach well. Thymidine incorporation was determined by scintillationcounting in triplicates.

Colony formation in semi-solid agar. Anchorage independent growth (theability of cells to form colonies in semi-solid agar) is characteristicof malignant glial cells. 9 L cells were harvested with trypsin/EDTA andresuspended at 1.0×10⁴ cells/ml in growth medium containing 0.36% agar(Difco). Two ml of the cell suspension was added to 60 mm plates(Costar, Cambridge, Mass.) which were precoated with 4 ml of solid agar(0.9%). Phenylacetate was added to the agar at different concentrations(0, 1.25, 2.5, and 5 mM). In a second experiment, 9 L cells were grownfor 7 days in tissue culture containing 5 mM NaPA. The cells were thentransferred, as described, to agar plates without NaPA. Coloniescomposed of 30 or more cells were counted after 3 weeks.

9 L brain tumor inoculation and phenylacetate administration. Fisher 344rats (n=50) weighing 230-350 grams were anesthetized usingintraperitoneal (i.p.) Ketamine (90 mg/Kg, Fort Dodge Laboratories,Inc., Fort Dodge, Iowa) and Xylazine (10 mg/Kg, Mobay Corporation,Shawnee, Kans.) and placed in a stereotaxic apparatus (David KopfInstruments, Tujunga, Calif.). 4×10⁴ syngeneic 9 L gliosarcoma cells in5 μL (Hank's) balanced salt solution were injected into the deep whitematter (depth of inoculation-3.5 mm) of the right cerebral hemisphereusing a 10 μL Hamilton syringe connected to the manipulating arm of thestereotaxic apparatus. In 10 rats, phenylacetate was administered bycontinuous subcutaneous (s.c.) release of the drug using two 2ML2osmotic pumps release rate of 5 μl/hr for 14 days (Alza Corporation,Palo Alto, Calif.). On the day of tumor inoculation the pumps wereimplanted in the subcutaneous tissue of both flanks. The concentrationof the drug in the pumps was 650 mg/ml (total of 2600 mg for both pumps)for a daily dose of 550 mg/kg per rat. The minipumps were replaced after14 days for a total treatment of 28 days. Fifteen additional ratsreceived NaPA, as described, starting 7 days after intracerebralinoculation of the tumor. In these rats, an additional daily injectionof NaPA (300 mg/kg, i.p.) was given for 28 days. Control rats (n=25)received continuous saline from two s.c. 2ML2 osmotic pumps.Perioperative penicillin (100,000 u/kg, i.m.) was given to all ratsbefore implantation of the minipumps. Survival was recorded in eachgroup. Three rats treated for established tumors and two control ratswere sacrificed 7 days after initiation of NaPA (14 days after tumorinoculation). These were used for electron microscopic studies oftreated tumors, in vivo proliferation assays, and measurement of NaPAlevels in the serum and CSF. Peripheral organs (heart, lung, spleen,liver, kidney, bowel, adrenal, and gonads) were harvested and subjectedfor a routine histological examination. Brain specimens were sectionedand stained for routine hematoxylin and eosin (H&E) and myelin stains(Luxol-fast blue) for evidence of drug-related toxicity.

Electron microscopy. Animals were sacrificed by intracardiac perfusionwith 1% paraformaldehyde and 2.5% gluteraldehyde in 0.1M sodiumcacodylate buffer at pH 7.4. Two hours later the fixed brains werewashed in buffer and sliced into 1 mm thick coronal sections. The areascontaining tumors were further dissected into 1 mm³ cubes, post-fixedwith 2% osmium tetroxide in 0.1M sodium cacodylate buffer for 2 hours,washed in buffer, mordanted en block with 1% uranyl acetate at pH 5overnight, then washed, dehydrated and embedded in Epon. Thin sectionswere cut at several levels into each block to ensure greater sampling.Electron micrographs of tumor cells were taken at random for morphology.

In vivo proliferation assay. One NaPA-treated and one saline-treated ratreceived an i.p. injection of 9 mg/3 ml of BrdU (Amersham, Ill.) 14 daysafter tumor inoculation and 7 days after initiation of treatment. Twohours later the rats were sacrificed and the brains were removed andsectioned. Mouse anti-BrdU monoclonal antibodies were used forimmunostaining of the tissues which were then counterstained withhematoxylin. Tumor cells in 10 high-power fields were enumerated in eachtumor specimen and the percent of positively staining cells (indicatingincorporation of BrdU during active cell division) was recorded.

Measurement of NaPA levels in serum and CSF. Three NaPA-treated and 2saline-treated rats were sacrificed after 7 days of combined s.c. andi.p. NaPA or saline administration. Blood was drawn from the heart andCSF was aspirated from the cisterna magna. Due to volume limitations ofCSF, pooled serum and CSF samples were assessed in a similar fashion.Protein extraction of a 200 μl aliquot of biological fluid was carriedout with 100 μl of a 10% perchloric acid solution. 150 μl of supernatewas neutralized with 25 μl of 20% potassium bicarbonate and centrifuged.125 μl of supernate was then pipetted into sampling tubes.Chromatography was performed on a Gilson 715 HPLC system using a 30 cmWaters C18 column (i.d. 3.9 mm) at 60° C. A 75 μl injectate was elutedwith an acetonitrile/water gradient ranging from 5 to 30% over 20minutes and flowing at 1 ml/min. UV-monitoring was performed at awavelength of 20 nm. Elution time for phenylacetate was 14.8 minutes.

Statistical analysis. The Chi-square test was used to compareproportions of BrdU-positive cells. The Mantel-Haenzel test was used tocompare survival between NaPA-treated and saline-treated rats in thesurvival experiments.

In Vitro Results

In vitro Effect of NaPA on cell proliferation and anchorage dependency.Treatment of 9 L cells with NaPA for 5 days resulted in dose-dependentdecrease in cell number with IC₅₀ at 6.0±0.5 mM. This was accompaniedwith a decrease in tritiated-thymidine incorporation (FIG. 8). Inaddition, phenylacetate induced a dose-dependent restoration ofanchorage dependency, indicating a reversion of the malignant phenotype(Table 19). 9 L cells that were exposed to NaPA for 7 days beforeplating in agar (not containing NaPA) still showed >40% inhibition incolony formation (Table 19).

                  TABLE 19    ______________________________________    Phenylacetate Inhibits    Anchorage-Independent Growth of 9L Gliosarcoma Cells    Treatment  PA in       Colony Formation    in Culture Agar (mM)   # Colonies                                    % Inhibition    ______________________________________    none       0           628 ± 50                                    --    none       5            8 ± 4                                    98.7               2.5         111 ± 13                                    82.4               1.25        326 ± 20                                    48.0    .sup.a Phenylacetate               0           375 ± 25                                    40.3    ______________________________________     .sup.a 9L cells were treated with 5 mM phenylacetate in culture for 7 day     before being plated on soft agar.

In Vivo Studies

In vivo proliferation assay and electron microscopy findings. Treatmentof established brain tumors with NaPA resulted in a significant decreasein the rate of proliferation. 285 of 1283 treated tumor cells stainedfor BrdU compared to 429 of 1347 saline-treated tumor cells (mitoticindex of 0.22 in NaPA-treated vs. 0.33 in saline-treated tumors;p<0.0001).

Electron microscopy of these tumors showed a striking abundance ofwell-organized rough endoplasmic reticulum in the NaPA-treated tumorcells indicating a higher degree of cell differentiation Ghadially F. N.Endoplasmic reticulum and ribosomes in cell differentiation andneoplasia. In: eds. Ultrastructural Pathology of the Cell and Matrix.Third, London:Buttorworths; 1992:450-454!. By contrast, untreated tumorsgenerally had scant rough endoplasmic reticulum and numerouspolyribosomes, which are characteristics of highly malignant cells.

In addition, mitotic cells were more frequently found in untreatedtumors.

Serum and CSF levels of NaPA. Assays of pooled serum and CSF from 3treated and 2 control rats, obtained after 7 days of combined s.c. andi.p. NaPA (total daily dose of 850 mg/kg) or saline administration,revealed a mean phenylacetate level of 2.45 mM in the serum and 3.1 mMin the CSF. No phenylacetate was detected in the serum of CSF samplesfrom saline-treated rats.

Survival Experiments

Simultaneous tumor inoculation and administration of NaPA. Seven of 10NaPA-treated rats survived for >90 days after tumor inoculation whenNaPA was administered for 4 weeks starting on the day of tumorinoculation. Nine of 10 control rats died within 34 days after tumorinoculation (p<0.01, Mantel-Haenzel test) (FIG. 9).

Treatment of established tumors with NaPA. Five of 12 rats treated withs.c. and i.p. NaPA for 4 weeks (starting 7 days after tumor inoculation)are still alive 50 days after tumor inoculation, while 12 of 13saline-treated rats died by day 36 (p<0.03, Mantel-Haenzel test) (FIG.10).

Toxicity. No adverse effects of NaPA treatment were detected in anytreated rats. Histological evaluation of the major peripheral organs andnon-tumoral brain showed no abnormalities.

Discussion. Phenylacetate induced a potent cytostatic and antitumoreffect in the in vitro and in vivo brain tumor models used in thesestudies. This effect extended beyond the duration of drugadministration, indicated by the long-term survival and apparent cure ofrats which received NaPA either simultaneously with tumor inoculation orafter tumors were established. This extended effect of NaPA shows thatthe malignant phenotype of treated tumor cells reverted, perhapsirreversibly in some animals, to one that was more benign anddifferentiated. Anchorage independence, i.e., the ability of cells toform colonies in semi-solid agar, is characteristic of malignant gliomacells. Phenylacetate caused a dose-dependent restoration of anchoragedependency, indicating reversion of the glioma cells to a non-malignantphenotype. More than 80% inhibition of colony formation was achieved atNaPA concentration in the agar plate of 2.5 mM, similar to the serum andCSF levels measured in treated rats. In addition, after one week ofexposure to NaPA, more than 40% of tumor cells maintained a benigngrowth pattern despite the absence of NaPA in the agar plates (Table19). A significant in vivo indicator of cell differentiation wasobserved in our study in the subcellular organelles of treated braintumor cells. The disorganized cytoplasmic polyribosomes in thesaline-treated tumor cells were transformed by NaPA to a hyperplastic,well organized, rough endoplasmic reticulum. The endoplasmic reticulumis a highly specialized structure that performs many distinct functions.Hence a well-developed endoplasmic reticulum represents celldifferentiation and functional activity. An inverse relationship hasbeen noted between the amount of rough endoplasmic reticulum and thegrowth rate and degree of malignancy of tumors Ghadially F. N.Diagnostic Electron Microscopy of Tumours. eds. 2. London:Butterworth;1985!. The numerous polyribosomes in the untreated tumor cellscorrelated well with the number of mitoses seen by light microscopy andwere confirmed by the BrdU proliferation assay. These changes underscorethe differentiating effect of NaPA on the malignant glial cells andcorrelate with the in vivo decrease in cell proliferation and extendedsurvival that occurred in treated animals with brain tumors.

Therapeutic blood and CSF NaPA levels were reached in the treated rats.The high CSF levels indicate good penetration of NaPA into the centralnervous system and into the developing tumor. The doses used are wellbelow the known toxic levels of NaPA in children with inborn errors ofurea synthesis (2.5 g/kg/d) or rats (1.6 g/kg/d) and indicate that NaPAcan be given safely at a higher doses, possibly with enhancement ofantitumor efficacy. These data indicate that phenylacetate, given torats at a non-toxic dose, has a profound effect on tumor growthregulation and cell maturation.

Example 9

Suppression of 5-Aza-2'-deoxycytidine induced carcinogenesis

Differentiation inducers selected for their low cytotoxic and genotoxicpotential could be of major value in chemoprevention and maintenancetherapy. Specifically, the ability of phenylacetate to preventcarcinogenesis by the chemotherapeutic hypomethylating drug,5-aza-2'-deoxycytidine (5AzadC), was tested in vitro and in mice.Transient exposure of immortalized, but non-tumorigenic ras-transformed4C8 fibroblasts to 5AzadC resulted in neoplastic transformationmanifested by loss of contact inhibition of growth, acquiredinvasiveness, and tumorigenicity in athymic mice. The latter wasassociated with increased ras expression and a decline in collagenbiosynthesis. These profound phenotypic and molecular changes wereprevented by a simultaneous treatment with phenylacetate. Protectionfrom 5AzadC carcinogenesis by phenylacetate was: (a) highly efficientdespite DNA hypomethylation by both drugs; (b) free of cytotoxic andgenotoxic effects; (c) stable after treatment was discontinued, and; (d)reproducible in vivo. Whereas athymic mice bearing 4C8 cells developedfibrosarcomas following a single i.p. injection with 5AzadC, tumordevelopment was significantly inhibited by systemic treatment withnontoxic doses of phenylacetate. Phenylacetate and its precursorsuitable for oral administration, phenylbutyrate, may thus represent anew class of chemopreventive agents, the efficacy and safety of whichshould be further evaluated.

The multi-step nature of neoplastic transformation makes this diseaseprocess amendable to chemopreventive intervention. Several agents havebeen shown to inhibit carcinogenesis and thereby prevent the developmentof primary or secondary cancers Kelloff, G. J., C. W. Boone, W. F.,Malone, and V. E. Steele. 1992. Chemoprevention clinical trials.Mutation Res., 267:291-295; Weinstein, B. I. 1991. Cancer prevention:Recent progress and future opportunities. Cancer Res., 51:5080s-5085s;Wattenberg, L. W. Inhibition of carcinogenesis by naturally occurringand synthetic compounds. In: Y. Kuroda, D. M. Shankel and M. D. Waters(eds), Antimutagenesis and Anticarcinogenesis, Mechanisms II,pp.155-166. New York: Plenum Publishing Corp., 1990; Sporn, M. B., andD. L. Newton. 1979. Chemoprevention of cancer and retinoids. Fed. Proc.38:2528-2534!. Of major interest are natural products and their analogs,including vitamins (A, B12, C, D3, and E), retinoids, and terpenes.These agents can suppress neoplastic transformation subsequent to acarcinogenic insult by regulating cell growth and differentiation. Onesuch growth regulator is phenylacetate.

The efficacy of phenylacetate as a chemopreventive agent was testedusing in vitro and in vivo models of 5AzadC-induced carcinogenesis.Despite the promise of 5AzadC in the treatment of cancer and ofbeta-chain hemoglobinopathies, its clinical applications have beenhindered by concerns regarding carcinogenic potential. The model used inthe present studies involved premalignant murine fibroblasts (cell lines4C8 and PR4), which express a transcriptionally activated c-Ha-rasprotooncogene. These non-tumorigenic cells are highly susceptible tomalignant conversion by pharmacological doses of 5AzadC. However,Phenylacetate can protect such vulnerable cells from 5AzadC-inducedcarcinogenesis both in culture and in mice.

Cell Cultures and Reagents

The subclones of mouse NIH 3T3 fibroblasts, PR4N and 4C8-A10 (designatedhere PR4 and 4C8) have been previously described Wilson, V. L., R. A.Smith, H. Autrup, H. Krokan, D. E. Musci, N-N-T. Le, J. Longoria, D.Ziska, and C. C. Harris. 1986. Genomic 5-methylcytosine determination by³² P-postlabeling analysis. Anal. Biochem., 152:275-284; Dugaiczyk, A.,J. J. Haron, E. M. Ston, O. E. Dennison, K. N. Rothblum, and R. J.Schwartz. 1983. Cloning and sequencing of a deoxyribonucleic acid copyof glyceraldehyde-3-phosphate dehydrogenase messenger ribonucleic acidisolated from chicken muscle. Biochem. 22:1605-1613!. Both cell linesare phenotypic revertants isolated from LTR/c-Ha-ras¹ -transformed 3T3cells after long-term treatment with murine interferon α/β. Cultureswere maintained in Dulbecco's modified Eagle's medium (DMEM)supplemented with 10% heat inactivated fetal calf serum (Gibco) andantibiotics. The sodium salts of phenylacetic and phenylbutyric acids(Elan Pharmaceutical Corporation) were dissolved in distilled water.5AzadC (Sigma St. Louis Mo.) was dissolved in phosphate buffered saline(PBS) and stored in aliquots at -20° C. until use. Exposure of 5AzadC todirect light was avoided at all times to prevent drug hydrolysis.

Treatments with 5AzadC

For treatment in culture, cells were plated at 1-2×10⁵ cells in 100 mmdishes and the drugs added to the growth medium at 20 and 48 hrs later.The cells were subsequently subcultured in the absence of the nucleosideanalogs and observed for phenotypic alterations. For in vivo treatmentwith 5AzadC, 6-9 week-old female athymic nude mice (Division of CancerTreatment, NCI Animal Program, Frederick Cancer Research Facility) wereinoculated subcutaneously (s.c.) with 0.5×10⁶ cells. Twenty four hourslater 400 μg of freshly prepared 5AzadC in 200 μl of PBS wasadministered intraperitoneally (i.p.) into each animal (approximately 20mg/kg). Systemic treatment with NaPA is described in the text.

Growth on Matrigel

The ability of cells to degrade and cross tissue barriers was assessedby a qualitative in vitro invasion assay that utilize matrigel, areconstituted basement membrane (Collaborative Research). Cells wereexposed for 48 hrs in T.C. plastic dishes with 5AzadC alone or incombination with NaPA. NaPA treatment continued for additional 1-2weeks. Cells were then replated (at 5×10⁴ per point) onto 16 mm dishes(Costar, Cambridge, Mass.), which were previously coated with 250 μl ofmatrigel (10 mg/ml). NaPA was either added to the dishes or omitted inorder to determine the reversibility of effect. Net-like formationcharacteristic of invasive cells occurred within 12 hours; invasion intothe matrigel was evident after 6-9 days.

Tumor Formation in Athymic Mice

Cells were injected s.c. (5×10⁵ cells per site) into 4-6 week old femaleathymic nude mice (Division of Cancer Treatment, NCI animal Program,Frederick Cancer Research Facility). The number, size, and weight oftumors were recorded after 3-4 weeks. For histological examination,tumors were excised, fixed in Bouin's solution (picric acid: 37%formaldehyde: glacial acetic acid, 15:5:1 vol/vol), and stained withH&E.

Measurement of DNA Methylation

To determine the 5-methylcytosine content, samples of cultures weretaken 24 hours after the second 5AzadC treatment. The cell pellets werelysed in 0.5% SDS, 0.1M NaCl, 10 mM EDTA pH 8.0, added with 400 μg/ml ofproteinase K (Boehringer Mannheim), and stored at -70° C. until DNAisolation and analysis. The content of methylated/unmethylated cytosineresidues in the cellular DNA was measured by a ³² P-postlabelingtechnique as previously described.

Northern Blot Analysis and DNA Probes

Cytoplasmic RNA was extracted from exponentially growing cells andseparated by electrophoresis in 1.2% agarose-formaldehyde gels. RNApreparation, blotting onto nylon membranes (Schleicher and Schuell),hybridization with radiolabeled DNA probes, and autoradiography wereperformed as described Rimoldi, D., V. Srikantan, V. L. Wilson, R. H.Bassin, and D. Samid. 1991. Increased sensitivity of nontumorigenicfibroblasts expressing ras or myc oncogenes to malignant transformationinduced by 5-aza-2'-deoxycytidine. Cancer Res., 51:324-330!. The DNAprobes included: 6.2 kb EcoRI fragment of v-Ki-ras, 2.9 kb SacI fragmentof the human c-Ha-ras¹ gene, and a BamHI 4.5 kb fragment of the c-mycgene. Glyceraldehyde phosphate dehydrogenase cDNA Dugaiczyk, A., J. J.Haron, E. M. Ston, O. E. Dennison, K. N. Rothblum, and R. J. Schwartz.1983. Cloning and sequencing of a deoxyribonucleic acid copy ofglyceraldehyde-3-phosphate dehydrogenase messenger ribonucleic acidisolated from chicken muscle. Biochem. 22:1605-1613! was provided by M.A. Tainsky (University of Texas, Houston), and a mouse transin cDNA byG. T. Bowden (University of Arizona, Tucson). The cDNA probe for mousehistocompatibility class I antigens was a gift from G. Jay (NIH,Bethesda). Radiolabeled probes were prepared with ³² P!dCTP (NEN) usinga random primed DNA labeling kit (Boehringer Mannheim, Germany).

In Vitro Carcinogenesis Induced by 5AzadC and Its Prevention byPhenylacetate

Untreated 4C8 and PR4 formed contact-inhibited monolayers composed ofepithelial-like cells. In agreement with previous observations,transient exposure of these cultures to 0.1 uM 5AzadC during logarithmicphase of growth resulted in rapid and massive neoplastic transformation.Within one week of 5AzadC treatment, the great majority of the cellpopulation became refractile and spindly in shape, and formedmultilayered cultures with increased saturation densities (Table 7),indicative of loss of contact inhibition of growth. These phenotypicchanges could be prevented by the addition of 5-10 mM NaPA (Table 7).Several different regimens of NaPA treatment were found to be similarlyeffective. These included: (a) pre-treatment with NaPA, starting one dayprior to the addition of 5AzadC; (b) simultaneous exposure to bothdrugs, and; (c) addition of NaPA one day after 5AzadC. In all cases,cells were subsequently subjected to continuous treatment with NaPA forat least one week. Cells cultured under these conditions, like thosetreated with NaPA alone, formed contact-inhibited monolayers resemblinguntreated controls. These cells maintained the benign growth pattern forat least three weeks after NaPA treatment was discontinued.

That NaPA prevents neoplastic transformation was further indicated bythe inability of cells to invade reconstituted basement membranes(matrigel), and form tumors in athymic mice. When plated onto matrigel,5AzadC-transformed 4C8 and PR4 cells developed net-like structurescharacteristic of highly malignant cells, and eventually degraded theextracellular matrix components. In marked contrast, NaPA-treatedcultures formed small, non-invasive colonies on top of the matrigel, aspreviously observed with normal fibroblasts. Untreated parental cellsexhibited an intermediate phenotype, as their colonies were slow growingand non-invasive, yet irregular in shape possibly due to increased cellmotility. The chemopreventive effect of phenylacetate could be mimickedby its precursor, phenylbutyrate. Cells exposed to 5AzadC in thepresence of sodium phenylbutyrate (NaPB, 1.5-3 mM) maintained contactinhibited growth and exhibited a benign phenotype when placed ontomatrigel (Table 7).

                  TABLE 7    ______________________________________    Effect of 5AzadC and NaPA on DNA Methylation                    DNA Methylation    Cells   Treatment.sup.a                          % 5mC.sup.b                                    % of Control    ______________________________________    4C8     none          3.49 ± 0.06                                    100            5AzadC        1.52 ± 0.27                                    43            NaPA          2.22 ± 0.10                                    63            5AzadC + NaPA 1.62 ± 0.18                                    46    PR4     none          2.72 ± 0.16                                    100            5AzadC        1.11 ± 0.22                                    41            NaPA          1.25 ± 0.08                                    46            5AzadC + NaPA 1.06 ± 0.11                                    39    ______________________________________     .sup.a Cells were treated with 0.1 uM 5AzadC and/or 10 mM NaPA and the     percentage of 5mC was determined as described in "Materials and Methods".     .sup.b Data indicate the mean ± S.D. (n = 4) of two experiments.

The in vitro growth characteristics of cells correlated with theirbehavior in athymic mice. 5AzadC-treated 4C8 cells developed rapidlygrowing fibrosarcomas within 2 weeks of s.c. transplantation into mice.Consistent with their behavior in vitro, the parental cells were farless aggressive, forming small lesions after 3-4 weeks in three of eightrecipient animals. However, no tumors developed in animals injected with4C8 cells that had been pre-treated for one week in culture with thecombination of 5AzadC and NaPA (Table 7). There was also no tumorformation in mice injected with 4C8 treated with NaPA alone. Thereforeit follows that NaPA induced phenotypic reversion of the premalignantfibroblasts and prevented their malignant conversion by the cytosineanalog.

Modulation of Gene Expression by NaPA

The NIH 3T3-derived cells lines, 4C8 and PR4, carry an LTR-activatedc-Ha-ras protooncogene. Northern blot analysis of 5AzadC-treated 4C8revealed a significant increase in ras mRNA levels and a decline in thedifferentiation marker, collagen a (type I) transcripts. No such changesin gene expression occurred in cultures to which NaPA was added.Withdrawal of NaPA after one week of continuous treatment did not causerestoration of ras expression, confirming that the therapeutic benefitof NaPA is stable in the absence of further treatment.

Effect of Phenylacetate and 5AzadC on DNA methylation

5AzadC is a potent inhibitor of DNA methylation, an epigenetic mechanismimplicated in the control of gene expression and cell phenotype.Hypomethylation may underlay the therapeutic effect of 5AzadC in cancerand in severe inborn anemias Momparler, R. L., G. E. Rivard, and M.Gyger. 1985. Clinical trial on 5-aza-2'-deoxycytidine in patients withacute leukemia. Pharmac. Ther., 30:277-286; Stamatoyannopoulos, J. A.,and A. W. Nienhuis. 1992. Therapeutic approaches to hemoglobin switchingin treatment of hemoglobinopathies. Annu. Rev. Med., 43:497-521; Ley, T.J., J. DeSimone, N. P. Anagnou, G. H. Keller, R. K. Humphries, P. H.Turner, P. H., N. S. Young, P. Heller, and A. W. Nienhuis. 1982.5-Azacytidine selectively increases gamma-globin synthesis in a patientwith beta⁺ thalassemia. N. Engl. J. Med. 307:1469-1475!. However,changes in DNA methylation could also be responsible for itscarcinogenic potential. It was of interest therefore to determine thedegree of DNA methylation in cells protected by phenylacetate. As wouldbe expected, 5AzadC caused a significant decrease in the content of5-methylcytosine (5 mC) (Table 6). There was, however, a comparabledecline in 5 mC in cells treated with 5AzadC in combination with NaPA,as well as in those treated with NaPA alone (Table 6).

                  TABLE 6    ______________________________________    In vitro Prevention by Phenylacetate of 5AzadC-Induced Carcinogenesis            Saturation        Tumorigenicity in Mice.sup.c    Cell      Density.sup.a                          Invas-         Tumor    Treatment (cells/cm.sup.2 × 10.sup.-5)                          iveness.sup.b                                  Incidence                                         Size (mm)    ______________________________________    None      3.9         -       3/8    1.0 (0.5-2)    5AzadC    7.0         +       8/8    11.5 (4-19)    5AzadC + NaPA              1.6         -       0/8    0    5AzadC + NaPB              1.1         -       ND    NaPA      ND          -       0/8    0    NaPB      1.3         -       ND    ______________________________________     .sup.a Cell were treated simultaneously with the indicated drugs and kept     in culture for 5 days post confluency at which time they were detached an     counted. Exposure to 5AzadC was transient as described in Materials and     Methods, while treatment with NaPA and NaPB continued throughout the     experiment. Similar results were obtained when NaPA treatment was     initiated one day prior or after cell exposure to 5AzadC (data not shown)     .sup.b Cells were plated on top of a matrigel layer and observed for     malignant growth pattern, i.e., development of characteristic processes     and degradation of the reconstituted basement membrane and invasion     towards the plastic surface below.     .sup.c Cells pretreated in culture were injected s.c (5 × 10.sup.5     cells per site) into 2 month old female athymic nude mice. Results     determined after 3 weeks indicate tumor incidence (tumor bearing, injecte     animals) and size. The values of tumor size are mean (range). ND = not     determined.

In Vivo Chemoprevention by NaPA

To determine the efficacy of NaPA in vivo, studies were extended toinclude an animal model involving athymic mice bearing thenon-tumorigenic 4C8 cells transplanted subcutaneously. A single i.p.injection of mice with 5AzadC (20 mg/kg) resulted in tumor developmentat the site of 4C8 cell inoculation. However, when mice were pre-treatedwith NaPA 1.5 hr prior to 5AzadC injection, and NaPA treatment continuedfor 22 days thereafter, the incidence of tumor formation wassignificantly decreased (Table 8). There were no adverse effectsassociated with NaPA treatment as indicated by animal weight andbehavior. Further more, despite causing DNA hypomethylation NaPA did notinduce neoplastic transformation of transplanted 4C8 cells. Animalsprotected by NaPA either failed to develop tumors or formed slow-growinglesions at the site of 4C8 inoculation. The animal data is consistentwith the in vitro findings, indicating that NaPA can prevent5AzadC-induced neoplastic transformation without producing significanttoxicities.

                  TABLE 8    ______________________________________    In vivo Chemoprevention by Phenylacetate           Animal       Tumor Incidence.sup.b                                    Tumor Size.sup.c    Group  Treatment.sup.a                        positive/total                                    mean (range)    ______________________________________    A      PBS          0/4         0    B      NaPA         0/4         0    C      5AzadC + PBS 9/9         12 (2-29)    D      5AzadC + NaPA                         4/10        3 (0-10)    ______________________________________     .sup.a 4C8 cells (5 × 10.sup.5 per site) were transplanted s.c. int     athymic mice. The next day, the animals in were treated i.p. with 400     mg/kg NaPA, and 1.5 hr later with 20 mg/kg 5AzadC. NaPA treatment was     repeated at 4.5 hours following 5AzadC injection. Subsequent treatments     involved NaPA injections twice daily for 8 days, and once a day for     additional 2 weeks. PBS was used as a control.     .sup.b Data indicates tumor growth at 4 weeks after 5AzadC treatment.     Spontaneous tumors developed thereafter in control animals receiving PBS,     and subsequently in those treated with NaPA.     .sup.c Tumor diameter in millimeters.

There is considerable interest in the use of nontoxic differentiationinducers in cancer chemoprevention. Drug toxicity is particularlyimportant considering the overall health condition and variablelife-span of candidate populations, i.e., high-risk individuals andpatients in remission. The differentiation inducer phenylacetate canprevent 5AzadC-induced carcinogenesis both in vitro and in vivo whenused at nontoxic doses.

Chemoprevention can be accomplished by either blocking the "initiation"step of carcinogenesis (i.e., mutagenesis), or by suppressing"promotion" and progression to malignancy. The current studies, usingpremalignant cells with an activated ras oncogene as a model, examinedthe efficacy of phenylacetate as an anti-promotional drug. Other wellcharacterized chemopreventive agents that block promotion includevitamin A and its synthetic retinoids; like phenylacetate, thesecompounds are also regulators of cell growth and differentiation.

The current studies exploited in vitro and in vivo models involvingfibroblasts (designated 4C8 and PR4) that are highly vulnerable tomalignant conversion by the DNA hypomethylating agents 5AzadC and 5AzaC(16,17). Transient exposure of these cells to 5AzadC, either in cultureor in recipient athymic mice, caused rapid neoplastic transformation.Malignant conversion was associated with an increase in ras mRNA levelsand down-regulation of collagen type I expression, indicating loss ofcell differentiation. These profound biological and molecular changesbrought about by 5AzadC are prevented by a simultaneous treatment withnon-cytotoxic concentrations of phenylacetate and its precursor,phenylbutyrate. Phenylacetate's antitumor activity and lack of toxicitywere confirmed in athymic mice. In the in vivo model, mice bearing thesusceptible 4C8 cells transplanted s.c. were injected i.p. with 5AzadC.All mice so treated developed rapidly growing fibrosarcomas; however,the incidence of tumor formation was markedly reduced by systemictreatment with NaPA.

The mechanism by which NaPA prevented the 5AzadC induced malignantconversion is unclear. Like other chemopreventive agents that blockpromotion, phenylacetate may act by inducing cytostasis and tumormaturation. There is a growing body of evidence indicating thatphenylacetate can cause selective growth arrest and tumordifferentiation in vitro and in rodent models. In some cases, e.g.,promyelocytic leukemia, differentiation induced by phenylacetate waslinked to a decline in myc oncogene expression. In NaPA-treated 4C8,protection from de-differentiation (evidenced by growth characteristicsand collagen expression), was associated with inhibition of rasoverexpression. Down-regulation of oncogene expression may thus beresponsible in part for the chemopreventive activity of NaPA. Inaddition to affecting ras at the mRNA levels, phenylacetate, aninhibitor of the mevalonate pathway of cholesterol synthesis Castillo,M., J. Iglesias, M. F. Zafra, and E. Garcia-Peregrin. 1991. Inhibitionof chick brain cholesterolgenic enzymes by phenyl and phenolicderivatives of phenylalanine. Neurochem. Int., 18:171-174!, could alsoblock the post-translational modification of the ras-encoded protein,p21. Limonene, an inhibitor of p21 prenylation, is a chemopreventiveagent as well.

Phenylacetate blocked carcinogenesis by 5AzadC despite the decline in 5mC content. In fact, NaPA itself was found to inhibit DNA methylation;yet, in contrast to 5AzadC, NaPA was not carcinogenic. Correlationsbetween carcinogenic potential and DNA hypomethylating activities ofchemical agents have been previously documented in tissue culturemodels, and alterations in DNA 5 mC patterns were proposed to contributeand enhance the initiation of carcinogenesis. However, the present dataindicate that quantitative changes in DNA methylation alone are notsufficient to affect cell phenotype and thus, hypomethylating activityis not sufficient to induce the tumorigenic phenotype in these in vitroand animal models.

The selective induction of specific genes by intracellular factors andchemical agents subsequent to demethylation has been reported by severallaboratories. For example, an increase in human gamma-globin geneexpression in vitro was found to require activation byhexamethylenebisacetamide following treatment with 5AzaC Ley J. T., Y.L. Chiang, D. Haidaris, N. P. Anagnou V. L. Wilson, and W. F. Anderson.1984. DNA methylation and regulation of the human β-globin like genes inmouse erythroleukemia cells containing human chromosome 11. Proc. Natl.Acad. Sci. USA. 81:6618-6622!; demethylation of the gene by 5AzaC wasnot sufficient for gene expression. By contrast, phenylacetate andphenylbutyrate induced gamma-globin gene expression with subsequentaccumulation of fetal hemoglobin in cultured erythroid progenitors andin humans. In addition to affecting DNA methylation, NaPA and NaPB alsoactivate a nuclear receptor that functions as a transcriptional factor(the peroxisome proliferator receptor is discussed herein). Thus, onepossible explanation for the differences in carcinogenic opposingactivities between NaPA/NaPB and 5AzadC seen here may be the ability ofthe aromatic fatty acids to induce the expression of genes critical togrowth control. Phenylacetate and related compounds can possibly reversethe methylation-mediated state of repression of silent anti-oncogenes.The finding of DNA hypomethylation by NaPA in mammalian cells does notcome as a surprise in view of previous studies demonstrating that, atmillimolar concentrations, phenylacetate inhibits DNA methylation inplant. Interestingly, at such high concentrations, phenylacetate alsoinhibits plant tumor cell proliferation. Therefore, the effect ofphenylacetate on DNA methylation and its role in regulating growth anddifferentiation have been conserved in evolution.

The outcome of combining NaPA with 5AzadC (or 5AzaC) is of particularinterest. The cytosine analogs have been shown to benefit patients withsevere blood disorders such as leukemia, sickle cell anemia, andβ-thalassemia. There is now experimental data suggesting that 5AzadC maybe active also in some solid tumors, including malignant melanoma (Weberet al, submitted) and prostate carcinoma. Unfortunately, the clinicalapplication of 5AzadC has been limited by concerns regardingcarcinogenesis. The data indicate that NaPA can minimize thecarcinogenic risk, while both preserving and potentiating thetherapeutic effects of 5AzadC. Studies with human leukemic cells andwith erythroid progenitors derived from patients withβ-hemoglobinopathies revealed that NaPA can enhance the efficacy of5AzadC, causing superinduction fetal hemoglobin production. Moreover,the addition of NaPA/NaPB to nontoxic, yet sub-optimal concentrations of5AzadC, induced complete growth arrest and promoted apoptosis incultured hormone-refractory prostatic carcinoma cells (unpublisheddata).

It appears therefore that phenylacetate, a common amino acid derivative,may be of value as an antitumor and chemopreventive agent. NaPA, whichhas an unpleasant odor, can be substituted by its precursor, NaPB (or aderivative or analog of NaPB), for oral administration. Upon ingestionby humans, phenylbutyrate undergoes β-oxidation to phenylacetate. LikeNaPA, NaPB exhibits antitumor and chemopreventive activities inexperimental models, and both drugs already proved safe for long-termoral treatment of children with urea cycle disorders. More recentclinical studies involving adults with cancer have confirmed thatmillimolar plasma levels of phenylacetate and phenylbutyrate can beachieved with no significant adverse effects. NaPB/NaPA will benefithigh risk individuals predisposed to cancer development, be applied incombination with other anticancer therapeutics to enhance efficacy andminimize adverse effects, and perhaps be used in maintenance therapy toprevent disease relapse.

Example 10

HbF induction in K562 cells by NaPA and derivatives

The K562 erythroleukemia line serves as a model for inherited anemiasthat are associated with a genetic defect in the beta globin geneleading to severe β-chain hemoglobinopathies.

The results reported in Table 9 also show that there is a synergisticaffect when leukemia cells are exposed NaPA in combination withinterferon alpha, a known biological response modifier or with thechemotherapeutic drug hydroxyurea (HU).

                  TABLE 9    ______________________________________    Induction of Hemoglobulin Synthesis in Erythroleukemia K562 cells*                                      CELL                         POSITIVE CELLS                                      VIABILITY    TREATMENT  BENZIDINE (%)          (%)    ______________________________________    Control               1.8         >95    NaPA    0.8 mg/ml             6.0    1.6 mg/ml            17.1    Interferon 500       13.5    IU/ml    HU 100 uM            17.2    NaPA (0.8 mg/ml) +   40-42    HU or IFN    ______________________________________     *Results at seven days of treatment.

Analysis of gene transcripts showed accumulation of mRNA coding forgamma globin, the fetal form of globin. This was confirmed at theprotein level.

Using the erythroleukemia K562 cell line described above it was foundthat 4-hydroxyphenylacetate was as effective as NaPA in inducing fetalhemoglobin accumulation, but was less inhibitory to cell proliferation.In contrast, some other analogs such as 2,4- or3,5-dihydroxyphenylacetate were found to be highly toxic.

Example 11

PC3 and DU145 cells--NaPA as an antitumor agent

The effectiveness of NaPA as an antitumor agent was further evaluated ina variety of experimental models. Studies in depth were performed withtwo androgen-independent human prostate adenocarcinoma cell lines, PC3and DU145, established from bone and brain metastases, respectively, aswell as hormone responsive LNCaP cultures. NaPA treatment of theprostatic cells resulted in concentration-dependent growth arrest,accompanied by cellular swelling and accumulation of lipid that stainedpositive with Oil-Red O. The results of this study are shown in FIG. 11.As illustrated therein, an IC₅₀ for NaPA occurred at 600-800 pg/ml.Significantly higher doses were needed to affect the growth of activelyreplicating normal human FS4 skin fibroblasts or normal endothelialcells (IC₅₀ from 12-15 μM), indicating a selective cytostatic effect ofthe drug.

Example 12

PC3 cells--non-invasiveness after NaPA treatment

It is known that PC3 cells are invasive in vitro and metastatic inrecipient athymic mice. Albini, A. et al. A rapid in vitro assay forquantitating the invasive potential of tumor cells. Cancer Res.47:3239-3245 (1987)!. The invasiveness of PC3 cells which is indicativeof their malignant phenotype can be assessed by their ability to degradeand cross tissue barriers such as matrigel, a reconstituted basementmembrane. Untreated PC3 cells and PC3 cells treated with NaPA for 4 daysin culture were quantitatively analyzed in a modified Boyden chambercontaining a matrigel-coated filter with FS4 conditioned medium as achemoattractant. After 4 days of treatment with 800 μg/ml of NaPA inT.C. plastic dishes, 5×10⁴ cells were replated onto 16 mm dishes(Costar, Cambridge, Mass.) coated with 250 μl of matrigel 10 mg/ml.Controls showed the characteristic growth pattern of untreated cells,i.e, formation of net-like structures composed of actively replicatingcells which eventually degraded the matrigel and formed monolayers onthe plastic surface beneath. In contrast to the controls, the NaPAtreated cells formed isolated small colonies which resembled normalhuman FS4 cells 8 days after plating. The NaPA treated cells failed todegrade the matrigel barrier. The formation of small noninvasivecolonies on top of the matrigel is indicative of loss of malignantproperties following treatment. Results of the in vitro invasion assayscorrelate highly with the biological behavior of cells in vivo.

Example 13

PC3 cells--PAG treatment did not hinder invasiveness

PC3 cells treated with NaPA for one week in culture, in contrast tountreated cells or those treated with PAG, failed to form tumors whentransplanted s.c. into athymic mice. These results are shown in Table10.

                  TABLE 10    ______________________________________    Tumorigenicity of Prostatic PC3 Cells in Nude Mice    TREATMENT              Diameter   Weight    (mg/ml)    Incidence   (mm ± S.D.)                                      (mg ± S.D.)    ______________________________________    None       7/7         9 ± 3   285 ± 60    NaPA 0.8   1/7         2           50    PAG 0.8    3/4         8 ± 2   245 ± 35    ______________________________________

PC3 cells were pretreated for 1 week in culture and then injected (2×10⁵cells/animal) s.c. into 4-5 week-old female athymic nude mice. Theresults in Table 10 indicate the incidence of tumor bearinganimals/injected animals as well as tumor size measured as meandiameter±S.D. 8 weeks later.

Example 14

Phenylacetate in combination with suramin

To further substantiate the phenotypic changes observed in the NaPAtreated prostatic PC3 cells, Northern blot analysis revealed that NaPAinhibited the expression of collagenase type IV, one of the majormetalloproteases implicated in degradation of basement membranecomponents, tumor cell invasion, and metastasis. Furthermore, it wasfound that NaPA treated prostatic PC3 cells showed an increase in thelevel of HLA-A mRNA which codes for major histocompatibility class Iantigen known to affect tumor immunogenicity in vivo.

The malignant prostatic cell lines exhibit numerous abnormalities ingene expression, including increased production of autocrine tumorgrowth factor-β (TGF-β) and elevated activity of urokinase plasminogenactivator (uPA). Members of the TGF-β family have been implicated intumor growth control, angiogenesis, and immunosuppression. uPA, incontrast, is a serine protease involved in degradation of extracellularstroma and basal lamina structures, with the potential to facilitatetumor invasion and metastasis. It was of interest, therefore, to examinethe effect of NaPA on TGF-β and uPa expression in the prostatic tumorcells. Northern blot analysis of PC3 after 72 h treatment revealed adecrease in TGF-β2 mRNA levels; the effect was specific for TGF-β2 asthere was no change in the expression of TGF-β1. The decrease in TGF-β2was accompanied by approximately a twofold increase in the levels ofHLA-A3 mRNA, as previously observed in treated human leukemic HL-60cells.

Preliminary analysis of uPA transcript levels showed no significantchange after NaPA treatment. There was, however, a reductioncell-surface uPA activity. The hormone-refractory malignant PC3 andDU145 cells, but not the more indolent hormone-responsive LNCaP,displayed high cell-bound uPA activity. Because the parental PC3cultures are composed of highly heterogenous cell populations withrespect to uPA production, more homogeneous subclones were establishedby limiting dilutions and single-cell cloning. A subclone designatedPC3-1, which resembled the parental PC3 cells in its invasive capacityand surface-localized uPA activity (2.2±0.3×10⁻⁶ Plau units per cell),was chosen for further studies. After 3 d of treatment of PC3-1 withNaPA 5 mM there was over 50% reduction in cell-associated uPA activity;the effect was dose-dependent and reversible upon cessation oftreatment. Similar results were obtained with DU145 cells. Assayspecificity was confirmed by the fact that pretreatment of cells withneutralizing anti-human uPA monoclonal antibodies, or addition ofantibodies at the time of assay, blocked over 95% of theplasminogen-dependent proteolytic activity. Plasminogen-independentproteolysis constituted 30% of the maximal fibronectin degradingactivity, and was similar for both NaPA-treated cells and untreatedcontrols.

                  TABLE 11    ______________________________________    Malignant Melanoma A375    Treatment      Growth     Viability    (μg/ml)     (% of control)                              (%)    ______________________________________    None           100        >95    NaPA 400       63.3       >95    Suramin     38            78.3       >95     75            56.8       >95    150            38.6       92    300            26.6       82    NaPA (400)    + Suramin (38) 45.5       >95    + Suramin (75) 30.1       94    + Suramin (150)                   21.8       92    ______________________________________

                  TABLE 12    ______________________________________    Prostate Adenocarcinoma PC3    Treatment      Growth     Viability    (μg/ml)     (% of control)                              (%)    ______________________________________    None           100        >95    NaPA 800       59.6       >95    Suramin    75             58.5       nd    150            46.5       nd    300            31.0       nd    NaPA (800)    + Suramin (75) 24.2       90    + Suramin (150)                   10.9       64    ______________________________________

NaPa was found to significantly potentiate the therapeutic effect ofsuramin, the only experimental drug known to be active against prostatecancer.

However, drug toxicities have been a major concern. In agreement withprevious in vitro studies, we found that toxic doses of suramin (300μg/ml) were needed in order to achieve over 50% inhibition of prostaticDU145 cell growth. This cellular model was used to examine whether NaPAcould enhance the activity of suboptimal but less toxic doses ofsuramin. Results of this examination show that NaPA and suramin act inan additive manner to inhibit DU145 cell proliferation. Moreover,suramin was found to be significantly more active if added toglutamine-depleted medium. Despite significant differences in tumorsensitivities, there was complete growth arrest when DU145 and PC3 cellswere treated for 6 d with both NaPA and suramin in glutamine-depletedmedium, under conditions in which each treatment alone had only apartial effect. Similarly, Tables 11 and 12 show the effect of combinedNaPA and suramin treatment of malignant melanoma A375 cells and prostateadenocarcinoma PC3 cells.

It is known that a disease state characterized by the presence of benignhyperplastic lesions of the prostate exists as a separate disease entityand has been identified in many patients that progress to a diagnosis ofprostatic cancer. Based on the above, it is anticipated that NaPA, inaddition to being effective in the treatment of prostatic cancer, wouldbe effective in treating patients having benign hyperplastic prostaticlesions.

Further experiments demonstrated that NaPA appears to have broadantitumor activity affecting a wide spectrum of malignancies. Theexperimental data presented in Table 13 indicate that NaPA 0.4-0.8 mg/mlcaused about 50% inhibition of growth in treated adenocarcinoma of theprostate cell lines PC3 and DU145, melanoma A375 and SK MEL 28, lungadenocarcinoma H596 and H661, and astrocytoma U87, U373, and 343.Somewhat higher concentrations (1.0-1.5 mg/ml) were needed to cause asimilar inhibition of squamous cell carcinoma A431, breast tumor MCS-7,osteosarcoma KRIB, and fibrosarcoma V7T. Typically, NaPA treatment wasassociated with growth arrest, induction of differentiation markers,reduced invasiveness in vitro, and loss of tumorigenicity in nude mice.

                  TABLE 13    ______________________________________    Responses of Different Tumor Cell Lines to NaPA Treatment                          % Inhibition by    #         Tumor Cell Line                          NaPA 0.8 mg/ml.sup.a    ______________________________________    1         Melanoma              A375        ≧70              SK MEL 28   >50    2         Prostatic Ca.sup.b              PC3         ≧50              DU145       ≧50              LaNCoP      >50    3         Astrocytoma              U87         ≧50              U343        ≧50              U373        ≧50    4         Kaposi's Sarcoma                          ≦40              KS    5         Leukemia HL-60                          ≦40    6         Leukemia K562                          ≦30    7         Breast Cancer                          ≦30              MCF-7    8         Osteosarcoma              KRIB        ≦30              HOS         <20    9         Fibrosarcoma              V7T         ≦30              RS485       ≦30    10        Squamous Cancer                          <30              of Head & Neck              A431    ______________________________________     .sup.a Pharmacologically attainable concentration     .sup.b Carcinoma

Of major interest in Table 13 are the following:

# 1-3 Tumor cells show significant response i.e., ≧50% inhibition ofproliferation within one week of treatment. Cf. FIG. 15.

# 4 KS, an HIV-associated disorder, may be more dramatically affected byNaPA in humans, due to inhibition of HIV expression and of essentialgrowth factors released by infected lymphocytes.

# 5,6 The treated HL-60 promeyelocytic leukemic cells undergo terminaldifferentiation, a desirable outcome of chemotherapy. In the K562erythroleukemia, NaPA induced reversible erythroid differentiation withno significant growth arrest (<30%); thus the K562 data is of interestwith respect to treatment of certain anemias, not cancer.

Less attractive

# 7-10 For effective responses, the tumors may require much higher drugconcentrations if used alone.

Although some of the malignant cell lines seem more sensitive thanothers, all were significantly more affected by NaPA when compared tonormal or benign cells. For example, NaPA inhibited the growth ofmalignant osteosarcoma (KRIB) cells more so than benignosteosarcoma-derived HOS cells. A differential effect was seen also inras-transformed fribrosarcoma V7T, when compared to the parentalnon-tumorigenic NIH 3T3 cells. As to normal human cells, as much as 2-4mg/ml of NaPA were needed to cause a significant inhibition of growth toprimary human skin FS4 fibroblasts. It should be noted that thetreatment was not toxic to either the malignant or the normal cells.

The concentration range found to selectively suppress malignant growthcan be readily obtained in the clinical setting without causingsignificant side effects. Intravenous infusion of NaPA into humans at250-500 mg/kg/day which results in plasma levels of 600-800 μg/ml hasbeen found to be a well tolerated treatment. Cytotoxicity in tissueculture was observed when the NaPA concentration was as high as 3 mg/mlor higher.

Example 15

Phase I clinical trials

Patient Population. Patients were eligible for this study if they hadadvanced solid tumors for which conventional therapy had beenineffective, a Karnofsky performance status greater than 60%, normalhepatic transaminases and total bilirubin, a serum creatinine less than1.5 mg/dl, and normal leukocyte and platelet counts. All patients signedan informed consent document that had been approved by the NationalCancer Institute (NCI) Clinical Research Subpanel. Seventeen patients,16 men and 1 woman, with a median age of 57 years (range: 36-75) wereenrolled between January and June 1993. Disease distribution includedprogressive, metastatic, hormone-refractory prostate cancer (9patients), anaplastic astrocytoma or glioblastoma multiform (6patients), ganglioglioma (1 patient) and malignant pleural mesothelioma(1 patient).

Drug Preparation and Administration

Sodium phenylacetate for injection was prepared from bulk sodiumphenylacetate powder supplied by Elan Pharmaceutical Research Co.(Gainesville, Ga.). The finished injectable stock solution wasmanufactured by the Pharmaceutical Development Service, PharmacyDepartment, Clinical Center, NIH, in vials containing sodiumphenylacetate at a concentration of 500 mg/ml in sterile water forinjection, USP, with sodium hydroxide and/or hydrochloric acid added toadjust the pH to approximately 8.5. Doses of sodium phenylacetate to beinfused over 30 minutes to 2 hours were prepared in 150 ml of sterilewater for injection, USP. Doses of phenylacetate to be given over 24hours were prepared similarly to yield a total volume of 1,000 ml andwere administered using an infusion pump.

The protocol as originally designed delivered an i.v. bolus dose ofphenylacetate (150 mg/kg over 2 hours) on the first day of therapy, toallow for the estimation of pharmacokinetic parameters. This wasfollowed 24 hours later by a CIVI of the drug for the next 14 days.Cycles of two week drug infusions were repeated every 6 weeks. The rateof drug infusion was to be increased in sequential cohorts of at leastthree patients, and individual patients could escalate from one doselevel to the next with sequential cycles of therapy provided they hadexperienced no drug-related toxicity and their disease was stable orimproved.

The protocol underwent several modifications over the 6 month period.First, the size of the initial bolus dose was reduced from 150 to 60mg/kg i.v. and the bolus infusion duration from 2 hours to 30 minutes,after the first three patients were treated. This change resulted indrug concentrations optimal for estimating the drug's pharmacokinetics(vide infra) within a six hour time period. Second, after the non-linearnature of phenylacetate's pharmacokinetics was recognized (vide infra),the protocol was changed from a fixed dose escalation (dose levels 1 and2:150 and 250 mg/kg/day, respectively) to a concentration-guidedescalation trial (dose levels 3 and 4:200 and 400 μg/ml, respectively).In the latter format each patient was given an i.v. bolus dose ofphenylacetate (60 mg/kg over 30 minutes) one week prior to beginning a14 day CIVI of the drug. The patient-specific pharmacokinetic parametersestimated from the bolus dose were used to calculate an infusion ratethat would maintain the serum phenylacetate concentration at thetargeted level during the 14 day infusion. Serum drug concentrationswere measured weekly, prompting weekining reestimation of individualpharmacokinetics and dosage adjustment (adaptive control with feedback).

Sampling Schedule

With the initial 150 mg/kg i.v. bolus, blood samples were obtainedthrough a central venous catheter at the following timepoints calculatedfrom the beginning of the infusion: 0, 60, 115, 125, 135, 150, 165, 180,240, 360, 480, and 600 minutes. For the 60 mg/kg bolus given over 30minutes, blood sampling was performed at 0, 30, 60, 75, 90, 105, 120,150, 180, 270 and 390 minutes from the beginning of the infusion. Atdose levels 1 and 2, blood samples were obtained daily during the CIVI,while at dose levels 3 and 4, blood samples were obtained on days 1, 2,3, 8, 9 and 10 of the infusion. Twenty-four hour urine collections forthe determination of phenylacetate and phenylacetylglutamine excretionwere obtained on days 1, 7 and 14 of therapy. Sampling of the CSF wasperformed only if clinically indicated.

Determination of sodium phenylacetate and phenylacetylglutamine in serumand urine by high performance liquid chromatography (HPLC). Blood wasdrawn by venipuncture into a Vacutainere tube free of anticoagulant andwas then refrigerated. It was centrifuged at 1,200 g for 10 minutes in aSorvall® RT 6000D centrifuge (DuPont Co., Wilmington, Del.) at 4° C.Serum was then removed and stored in Nunc Cryotubes (Nunc Co., Denmark)at -70° C. until the day of analysis.

A standard curve was generated by adding known amounts of sodiumphenylacetate (Elan Pharmaceutical Research Co., Gainesville, Ga.) andphenylacetylglutamine (a gift from Dr. S. W. Brusilow, Johns HopkinsUniversity, Baltimore) to a commercial preparation of pooled serum(Baxter Healthcare Corporation, Deerfield, Ill.). The standard valuesspanned the expected range of serum concentrations: 0, 5, 10, 20, 50,100, 250, 500, 750 and 1,500 μg/ml.

Two hundred microliters of serum were pipetted into a 1.7 ml Eppendorfttube (PGC Scientifics, Gaithersburg, Md.). Protein extraction wascarried out by adding 100 μl of a 10% (v/v) solution of perchloric acid(Aldrich Chemical Co., Milwaukee, Wis.). The tube was vortexed and thencentrifuged at 4,500 g for 10 minutes. One hundred and fifty microlitersof supernatant were transferred to a new 1.7 ml Eppendorf tube and 25 μlof 20% KHCO₃ (w/v) was added to neutralize the solution. This wascentrifuged at 4,500 g for 10 minutes and 125 μl of supernatant weretransferred to an autosampler vial and maintained at 10° C. until HPLCinjection. Urine samples were processed in an identical manner after aninitial 1:10 dilution with water.

The HPLC system (Gilson Medical Electronics, Middleton, Wis.) wascomposed of two pumps (305 and 306), an 805 manometric module, an 811Cdynamic mixer, a 117 variable wavelength UV detector and a 231autosampler fitted with a 20 μl injection loop and cooled with a GreyLine model 1200 cooling device. The column was a Waters® (MilliporeCorporation, Milford, Mass.) C18 Nova-Pak, 3.9×300 mm, maintained at 60°C. with a Waters® temperature control module. The mobile phase solutionsconsisted of fifty microliter samples were auto-injected onto a 10 cmcation-ion exchange column integrated into a Beckman Model 6300 AminoAcid Analyzer (Beckman Instruments Inc., Palo Alto, Calif.). The solventflow rate (2:1 water/ninhydrin) was maintained constant at 0.5 ml/min.Column temperature was raised by 1.5° C. per minute to elute sarcosine,the internal standard. The column was regenerated with lithium hydroxideat 70° C. following each injection. Absorbance was measured at 570 nmand 440 nm following post-column color development with ninhydrin-RX(Beckman Instruments Inc., Palo Alto, Calif.) at 131° C. Beckman SystemGold software was used for data acquisition and data management.

Pharmacokinetic Methods

Initial estimates of V_(MAX) and K_(M) for phenylacetate were obtainedby generating Lineweaver-Burk plots from concentration versus timecurves following i.v. bolus doses. These initial parameter estimateswere refined by non-linear least squares fitting, using the Nelder-Meaditerative algorithm, as implemented in the Abbottbase® PharmacokineticSystems software package (Abbott Laboratories, Abbott Park, Ill.,version 1.0).

Statistical Methods

The Student's t-test was used to compare estimates of phenylacette'spharmacokinetic parameters derived from the Lineweaver-Burk plots withthose obtained using non-linear given set of dosing and concentrationdata was quantified by calculating the weighted sum of the errorssquared following non-linear least-squares fitting. The standarddeviation of the errors was modeled as a function of drug concentrationmultiplied by the coefficient of variation of the assay. Confidenceregions for the parameters were derived from the weighted sum of squaresin the model incorporating the induction parameters, and approximatesignificance levels for testing between the two models were calculatedusing the F distribution Draper, N. R., Smith H. Applied RegressionAnalysis. New York, John Wiley and Sons, p. 282, 1966!. The significancelevels of individual cycles were analyzed by the Spearman rankcorrelation method in an attempt to discern whether a relationshipexisted between time-dependent changes in drug clearance and dose.

Analytical Assay

The reverse phase HPLC assay allowed both serum phenylacetate andphenylacetylglutamine concentrations to be determined simultaneouslyfrom the same sample (see FIG. 15). The lower limit of detection forboth compounds in serum and urine was 5 μg/ml, based upon asignal-to-noise ratio of 5:1. The interassay CV for serum concentrationswas less than 6% within the range of 40 to 1,000 μg/ml. (Table 14). Thelower limit of detection for glutamine was 0.5 μg/ml, with an interassayCV that did not exceed 7%.

Model Specification and Initial Parameter Estimation

FIG. 13 shows representative concentration versus time curves forsimultaneously measured serum levels of phenylacetate andpheylacetylglutamine and plasma levels of glutamine following a 150mg/kg bolus dose of sodium phenylacetate. The post-infusion decline inserum phenylacetate concentration over time is linear when plotted on anon-logarithmic scale, consistent with saturable elimination kinetics.While useful for demonstrating a zero-order process, the 150 mg/kg boluswas inadequate for parameter estimation insofar as most of thephenylacetate concentrations obtained over the six-hour sampling periodwere above K_(m). In order to generate concentrations both above andbelow K_(m), the bolus was changed to 60 mg/kg i.v. over 30 minutes.Visual inspection of the concentration versus time curves followingthese boluses revealed no evidence of an initial distributive phase,suggesting that a single compartment, open non-linear model should beadequate to describe the drug's pharmacokinetics. Initial estimates(mean±SD) of K_(m) (90±30 μg/ml), V_(max) (26.0±10 mg/kg/hr) and Vd(22.4±6.8 L) were calculated in 13 patients using the Lineweaver-Burkequation. Refinement of these initial parameter estimates by non-linearleast squares fitting of the entire concentration versus time profilefor each bolus dose yielded the following estimates: K_(m) =105.1±44.5μg/ml, V_(max) =24.1±5.2 mg/kg/hr and Vd=19.2±3.3 L. The differencesbetween the two methods of estimation were not statistically different,as measured by the Student's t-test (p=0.89).

Induction of Phenylacetate Clearance

In some patients treated at dose levels 1 and 2, we observed a tendencyfor the serum phenylacetate concentration to decrease with time. Anexample of this phenomenon is shown in FIG. 11. Considering the 12cycles of therapy delivered at these levels, a comparison of the serumdrug concentration measured on day 2 of CIVI to that observed on day 11demonstrated a statistically significant decline in concentration withtime (Wilcoxon signed rank test, p=0.016). At dose levels 3 and 4,attempts at maintaining targeted serum phenylacetate concentrationsusing adaptive control with feedback led to variable rates of druginfusion over time, which precluded a simple comparison of drugconcentrations at the beginning and end of therapy.

Therefore all cycles of therapy were analyzed at all four dose levelsand compared with the performance of the single compartment non-linearmodel described above with the same model modified to allow V_(max) toincrease with time. The formula used to describe this increase was:

    V.sub.max (1)=V.sub.max,t=0 ×{1.0+ (IF-1.0)×(1.0-e.sup.IR×t)!}

wherein t is the time elapsed (in hours) since the initiation oftherapy, IF is an induction factor representing the maximum-foldincrease in V_(max) at infinite time and IR is a first order rateconstant (h⁻¹) describing the rate at which V_(max) increases over time.Each cycle of therapy (n=21) was evaluated by comparing the differencein the weighted sum of errors squared generated by non-linearleast-squares-fitting with each model. The significance of thedifference was evaluated using the F test (see statistical methods). In9 of the 21 cycles, allowing V_(max) to increase with time yielded animproved fit (induction parameters, mean±SD:IF=1.87±0.37,IR=0.0028±0.003 h⁻¹, p≦0.035). The Spearman rank correlation method didnot demonstrate a correlation between rate of drug administration andthe need to incorporate the two induction parameters into the model(rank correlation coefficient=0.39, p=0.084). The dose ratesadministered ranged from 450 to 1,850 mg/h.

Review of concomitantly administered medications revealed no associationbetween specific drugs and the occurrence of a time-dependent increasein phenylacetate clearance. In seven patients with primary CNS tumors,treatment with anticonvulsants always antedated the administration ofphenylacetate by months to years.

Mechanisms of Phenylacetate Clearance

As shown in FIG. 13, phenylacetate underwent rapid conversion tophenylacetylglutamine. In the three patients who received 150 mg/kg ofphenylacetate over 2 hours, the peak serum concentration ofphenylacetylglutamine (mean±SD) was 224 ±81 μg/ml, 325±72 minutespost-infusion. After 60 mg/kg boluses, the peak serumphenylacetylglutamine concentration was 104±33 μg/ml at 86±33 min. Theplasma glutamine concentration prior to bolus treatment withphenylacetate was 105±29 μg/ml (mean±SD, n=16), similar to reportedvalues in the literature for normal volunteers. The largest reduction incirculating plasma glutamine levels (46%) was observed in a patientreceiving a 150 mg/kg bolus.

The molar excretion of phenylacetylglutamine was determined from 24 hoururine collections. It accounted for 99±23% (n=18) of the dose ofphenylacetate administered over the same period of time. The recovery ofthe free, non-metabolized drug was only 1.5±2.4% of the totaladministered dose. A strong phenylacetate odor was detectable onpatients' clothes and on examiners'hands after physical examination.This suggests that phenylacetate may also be excreted to some extenttransdermally.

Distribution of Phenylacetate and Phenylacetylglutamine into the CSF

Clinical circumstances required evaluation of the cerebrospinal fluid intwo patients who had metastatic prostate cancer and were free of CNSmetastases. The first had reached steady-state phenylacetate andphenylacetylglutamine concentrations of 141 and 199 μg/ml, respectively,the corresponding simultaneous CSF concentrations were 74 and 5 μg/ml,respectively. At the time of simultaneous serum and CSF sampling, thesecond patient had been off therapy for 6 hours after having reached aserum concentration of phenylacetate of 1044 μg/ml. Measurements inserum and CSF were 781 versus 863 μg/ml for phenylacetate and 374 versus46 μg/ml for phenylacetylglutamine, respectively.

Clinical Toxicities

No toxicity was associated with bolus administration of the drug. Thehighest peak serum concentrations were measured after the 150 mg/kgbolus over 2 hours (533±94 μg/ml, mean±SD). Table 15 lists the averageserum phenylacetate concentrations per dose level. Although thoseachieved at dose levels 3 and 4 are close to their target, the largestandard deviations reflect our inability to maintain serumphenylacetate concentrations within the desired range, even when usingadaptive control with feedback.

Drug-related toxicity was clearly related to the serum phenylacetateconcentration. Three episodes of CNS toxicity, limited to confusion andlethargy and often proceeded by emesis, occurred in patients treated atdose levels 3 and 4. They were associated with drug concentrations of906, 1044 and 1285 μg/ml (mean: 950±300 μg/ml), respectively. Symptomswere completely resolved within 18 hours of terminating the druginfusion in all instances,

Antitumor Activity

Stabilization of PSA for more than 2 months was noted in 3 of the 9patients with prostate cancer treated at dose levels 2, 3 and 4 (meanphenylacetate concentration: 234±175 μg/ml). A fourth patientexperienced marked improvement in bone pain and was able to substitute anon-steroidal anti-inflammatory drug to his morphine regimen. Onepatient with glioblastoma multiform has had improvement in performancestatus (30% on Karnofsky's scale), intellectual function and expressiveaphasia of greater-than 5 months duration. Although no change in thesize of the tumor mass was noted, reduction in peritumoral edema wasdocumented by MRI.

Discussion

Previous descriptions of the pharmacokinetics of phenylacetate have beenfragmentary. Simell et al. reported the drug to have first orderelimination kinetics with a half-life of 4.2 hours following bolus doseadministration 9270 mg/kg) in children Simell, O, Sipila, I, Rajantie,J, Valle, D. L., and Brusilow, S. W. Waste nitrogen excretion via aminoacid acylation: benzoate and phenylacetate in lysinuric Proteinintolerance. Pediatr. Res., 20:1117-1121, 1986!. The failure torecognize the non-linear nature of phenylacetate pharmacokineticsprobably resulted from the smaller total doses given to these patientscompared to those given in our study. The saturable pharmacokinetics ofphenylacetate are consistent with an enzymatic process and ourcalculations from the 24 hour urinary excretion of phenylacetylglutamineconfirm that this is the major route of elimination. Evidence that drugclearance increases with time was derived from the comparison of druglevels on days 2 and 11 of the CIVI, adding another layer of complexityto the pharmacokinetics of phenylacetate. To explain this phenomenon,the potential role of concomitantly administered medications was firstconsidered, but failed to demonstrate any association. Analysis of a therelationship between an increase in drug clearance with time and therate of drug administration did not reach statistical significance andsuffered from the small number of cycles of therapy available foranalysis. It should also be noted that, relative to the 14 day periodover which it is assessed, V_(max) tended to increase slowly, with anaverage half-time calculated from the induction rate (IR) of 9.6 days.

As expected for such a small molecule, phenylacetate readily penetratesinto the CSF, which may explain the dose-limiting side-effects of thedrug, i.e., nausea, vomiting, sedation and confusion.

The results of Table 15 indicate that attempting to maintain serumphenylacetate concentrations at either 200 or 400 μg/ml using adaptivecontrol with feedback was problematic, with drug concentrations thatoften greatly exceed the level-specific targets. All patients whoexhibited CNS toxicity had serum phenylacetate concentrations in excessof 900 μg/ml. In the average patient, the drug must be infused at a rateequal to 75% of V_(max), in order to maintain a constant serumphenylacetate concentration of 400 μg/ml, which is four times greaterthan K_(m). Thus, the slightest error in the estimation of individualpharmacokinetics or in the rate of drug infusion results in largechanges in drug concentration. Phenylacetate was delivered by CIVI inorder to mimic the preclinical conditions that had demonstratedantitumor activity, namely, continuous exposure to concentrations above475 μg/ml for at least two weeks. Unfortunately, such concentrationscannot be practically maintained.

An alternative strategy is to deliver the drug by repeated shortinfusions. Our limited experience with the 150 mg/kg i.v. bolusessuggests that serum phenylacetate concentrations occurring transientlyabove 500 μg/ml are well tolerated. In addition, the time intervalbetween infusions allows some drug washout to occur, thereby minimizingdrug accumulation. A simulated regimen of 200 mg/kg q 12 h (1 hourinfusion) is presented in FIG. 16. The simulation assumes that thepharmacokinetic parameters determined from our 17 patients arerepresentative of the cancer population at large and that V_(max) doesnot change with time. It predicts that a wide range of peak drugconcentrations will be present. However, it is possible that these wouldbe sufficiently transient so as not to produce CNS toxicity and thetroughs not so prolonged as to abrogate the drug's antitumor activity.

                  TABLE 14    ______________________________________    PA Standard Curve Assay Variability    PA        CV           PAG     CV    (μg/ml)              (%)          (μg/ml)                                   (%)    ______________________________________    40        2.6          40      4.6    400       1.7          400     4.3    1000      3.4          1000    3.1    ______________________________________

                  TABLE 15    ______________________________________    PA and PAG Concentrations Per Dose Level During CIVI                           PA.sup.a  PAG.sup.a    Dose Level PA dose level                           (μg/ml)                                     (μg/ml)    ______________________________________    1          150 mg/kg/d  49 ± 19                                      90 ± 34    2          250 mg/kg/d 104 ± 40                                     150 ± 63    3          200 μg/ml                           178 ± 85                                     188 ± 55    4          400 μg/ml                            397 ± 244                                     306 ± 51    ______________________________________     .sup.a mean ± SD

Example 16

Effect of NaPA on differentiation of human neuroblastoma cells.

The ability of NaPA to promote the differentiation of humanneuroblastoma cells was studied, both alone and in combination withretinoic acid (RA), a known inducer of neuroblastoma differentiation andmaturation. In the LA-N-5 cell line, phenylacetate stimulated thedifferentiation of human neuroblastoma cells as evidenced bydose-dependent inhibition of cell proliferation, neurite outgrowth,increase acetylcholinesterase activity, and reduction of N-myc proteinlevels. Furthermore, NaPA and RA synergized in inducing LA-N-5differentiation in that combination treatment resulted in completecessation of cell growth along with morphologic and biochemical changesindicative of the loss of malignant properties. The combined effectsrepresent a strong differentiation response in neuroblastoma cells, bothas to number of responding cells and maturational level achieved.Transient transfection of LA-N-5 cells with a variety of CAT reportergene plasmids including constructs containing thyroid and RA responsiveregulatory elements have suggested that the pathways of action of NaPAand RA may intersect at the nuclear level through activation of commonresponse elements. The synergistic effects, thus, may be mediated by theability of NaPA to modulate the RA differentiation pathway so as toresult in altered transactivation of RA responsive regulatory elementsin relevant target genes. These in vitro antineoplastic effects wereobserved under drug concentrations achievable in humans withoutsignificant toxicities.

Section B: Phenylacetate and its Derivatives in the Treatment andPrevention of Aids

The etiology of human acquired immunodeficiency syndrome (AIDS) has beenlinked to the human immunodeficiency virus (HIV), which is capable ofselective infection and suppression of the host immune system. Thisimmune defect renders the human body susceptible to opportunisticinfections and cancer development, which are ultimately fatal. Thespread of HIV throughout the world is rapid, with no effectivetherapeutics on hand. It is suggested that NaPA, a nontoxic naturalcompound capable of glutamine depletion in vivo, can be used in thetreatment and prevention of AIDS.

HIV is a retrovirus. The production of retroviruses is dependent ontranscriptional activation by the long terminal repeat (LTR) element,and the availability of glutamine (Gln) for translational control.Experimental data obtained with chronically infected cultured cells andanimal models indicate that virus replication is specifically inhibitedin cells starved for glutamine, but not in those starved for other aminoacids (Gloger and Panet (1986); (J. Gen. Virol. 67:2207-2213) Robertsand McGregor, (1991), (J. Gen. Virol 72:2199-305). The results could notbe attributed to either an effect on cell cycle or a general inhibitionof protein synthesis.

The reason why glutamine depletion leads to virus suppression can beexplained as follows. Replication competent murine retroviruses containan amber termination codon at the junction of gag and pol genes, whichcan be recognized by amber suppressor tRNA^(Gln). Glutamine is thusessential for the readthrough of viral mRNA transcripts Yoshinaka et al.(1985); PNAS 82:1618-1622!. Reduction in glutamine concentrationsdisrupts viral mRNA translational readthrough and protein synthesis,with subsequent inhibition of viral assembly and secondary spread.Although human retroviruses are somewhat different from the murineviruses studied, it has been shown that reduction in the levels of ambersuppressor tRNA^(Gln) in human cells infected with HIV causes asignificant reduction in the synthesis of viral proteins Muller et al.Air Research and Human Retroviruses 4:279-286 (1988)!. Such data suggestthat agents which can lower glutamine levels in humans are likely tobenefit patients infected with HIV. NaPA may be such an agent, since itis known to conjugate to glutamine in humans with subsequent renewedexcretion of phenylacetylglutamine. Since NaPA also possesses antitumoractivities, the drug is likely to affect Kaposi's sarcomas, the tumorsfound in as many as 30% of all AIDS patients, as well as lymphomasassociated with AIDS.

Example 17

NaPA for treatment of AIDS related disorders

Evidence from experimental model systems in support of the abovehypotheses includes:

(a) Preliminary findings with cultured cells indicating that NaPA caninhibit expression of genes controlled by the retroviral LTR; (b) Whileanimal studies have been hindered by the fact that glutamine depletionby NaPA is limited to humans and higher primates, an acceptable animalmodel (other than primates) involves rodents treated with glutaminase.The expression of retroviral genes is under the control of the longterminal repeat (LTR) element; inhibition of LTR would preventtranscription and synthesis of viral proteins. To examine the effect ofNaPA on the retroviral LTR, V7T fibrosarcoma cells carrying anLTR-dependent Ha-ras oncogene were used as a model. Results of Northernblot analysis showed markedly reduced levels of the ras RNAtranscription in cells treated with NaaPA compared to RNA transcriptionlevels in untreated control cells. The results cannot be explained by ageneral effect on gene expression, as indicated by the increasedexpression of the cellular genes collagen and 2'-5' oligo adenylatesynthetase (2-5 ASyn). The latter are of particular interest sincecollagen is a marker of fibroblast differentiation, and 2-5 ASyn isassociated with growth control. Taken together, the data indicate theNAPA suppressed the activity of the retroviral LTR, while restoringgrowth control and differentiation to the host cells. Similarlydesirable changes might occur in HIV-infected monocytes and T4lymphocytes following systemic treatment of afflicted patients withNaPA. Glutaminase is a bacterial enzyme that causes reduction ofextracellular (and presumably intracellular) glutamine concentrations.Glutaminase treatment of viremic mice infected with Rouscher murineleukemia virus (RLV) inhibited retroviral replication and thedevelopment of splenomegaly, and significantly increased animal survivalRoberts and McGregor J. Gen. Virology 72:29-305 (1991)!. The efficacy ofglutaminase therapy compared favorably with AZT, the drug currently usedfor treatment of AIDS. The results are of particular interest since theRLV serves as a model in the search for anti-HIV drugs (Ruprecht et al.,1986). Unfortunately, however, glutamine depletion by glutaminase invivo is only transient due to development of neutralizing antibodies tothe enzyme. Once this occurs, viral replication can resume, eventuallykilling the host. NaPA, unlike the bacterial glutaminase, is a naturalcomponent of the human body, and thus is less likely to induce theproduction of neutralizing antibodies; (c) There is clinical evidencefor sustained reduction by NaPA of plasma glutamine concentrations. NaPAis currently being used for treatment of hyperammonemia associated withinborn disorders of urea metabolism. Clinical experience indicates thatlong-term treatment with NaPA effectively reduces glutamine levels. Suchtreatment is nontoxic and well tolerated even by newborns. Inconclusion, NaPA might benefit patients with HIV infection. NaPA couldinhibit viral replication through (among other mechanisms) inhibition ofLTR and depletion of glutamine, the amino acid required for appropriateprocessing of viral proteins. If NaPA proves to have anti-HIV activitiesin humans, it could be used to prevent disease progression inasymptomatic HIV-positive individuals. The lack of toxicity, easy oraladministration and relatively low cost uniquely qualify NaPA as achemopreventive drug. In fact, the drug is so well tolerated by humansthat treatment can start just a few hours after birth. In addition, NaPAcould be used (alone or in combination with other drugs) in treatment ofAIDS-associated disorders including opportunistic infections, HIVencephalopathy, and neoplasia.

Section C: Induction of Fetal Hemoglobin Synthesis in β-ChainHemoglobinopathy by Phenylacetate and its Derivatives

There is considerable interest in identifying nontoxic therapeuticagents for treatment of severe β-chain hemoglobinopathies. Employing thehuman leukemic K562 cell line as a model, we have explored the cellularresponses to NaPA, an amino acid derivative essentially nontoxic tohumans. Treatment of cultures with pharmacologically attainableconcentrations of NaPA resulted in time- and dose-dependent inhibitionof cell proliferation and caused an increase in hemoglobin production.Molecular analysis revealed accumulation of the fetal form of hemoglobin(HbF), which was associated with elevated steady-state levels of gammaglobin mRNA. All NaPA effects reversed upon cessation of treatment.Interestingly, addition of NaPA to other antitumor agents of clinicalinterest, i.e., 5-azacytidine and hydroxyurea, resulted insuperinduction of HbF biosynthesis. The results suggest that NaPA, anagent known to be well tolerated by newborns, could be used alone or incombination with other drugs for long-term treatment of some inbornblood disorders.

The pathophysiology of inherited blood disorders such as sickle cellanemia and severe β-thalassemias is based on genetic abnormalities inthe β-globin gene which result in deficient or absent β-globinsynthesis. The latter prevents the production of hemoglobin and resultsin ineffective red blood cell production and circulation. Recent dataindicate that pharmacological manipulation of the kinetics of cellgrowth and differentiation might have a beneficial effect in patientswith the β-chain hemoglobinopathies, due to the induction of fetalhemoglobin (HbF) synthesis. To date, several antitumor drugs including5-azacytidine (5AzaC), 5-aza-2'-deoxycytidine (5AzadC), hydroxyurea(HU), vinblastine, and arabinosylcytosine (ara-C) have been shown toincrease the production of HbF in experimental models Dover, Ann N.Y.Acad. Sci. 612:184-190 (199)!. Moreover, there is clinical evidence for5AzaC and HU activity in severe β-thalassemia and sickle cell anemia,respectively. However, concerns regarding toxic and potentialcarcinogenic effects of the prevailing antitumor drugs raise the need toidentify safe alternatives for long-term treatment of the inbornnonmalignant diseases. The accumulation of fetal hemoglobin in adults isthought to be due to changes in the kinetics of erythroiddifferentiation rather than a direct effect on the fetal globin genes.According to this hypothesis, other agents that can inducedifferentiation would also be expected to affect HbF production. Thefocus here is on the efficacy of a novel nontoxic differentiating agent,NaPA.

As discussed in Section A, Applicant's laboratory has found that NaPAcan also affect the maturation (i.e., differentiated state) of variousanimal and human cell types. The drug caused growth arrest and reversalof malignant properties in a variety of in vitro tumor models includingcell lines established from adenocarcinomas of the prostate and lung,malignant melanomas, and astrocytomas. Moreover, NaPA treatment wasassociated with adipocyte conversion in premalignant mesenchymal C3H10T1/2 cells, and granulocyte differentiation in promyelocytic leukemiaHL-60 cultures. Studies indicated that NaPA, in contrast to thechemotherapeutic differentiating drugs 5AzaC and 5AzadC, may be free ofadverse effects such as cytotoxicity and tumor progression.

Indeed, NaPA is well tolerated by humans as indicated by the vastclinical experience with NaPA in the treatment of hyperammonemia ininfants with inborn errors of ureagenesis. The clinical experienceindicates that acute or long-term treatment with high doses of NaPA isessentially free of adverse effects. The lack of toxicity and theability to induced cellular differentiation prompted Applicant toexamine the effect of NaPA on HbF expression.

Example 18

K562 cells--induction of HbF by treatment with NaPA

The experimental system involved the human leukemic K562 cells, whichcarry a nonfunctional β-globin gene, but produce low levels of the fetalgamma globin and of HbF. The K562 cell line was originally establishedfrom a patient with chronic myelogenous leukemia in the blast cellstransformation, and has since been extensively utilized as a model instudies of erythroid differentiation and regulation of the gamma globingene expression. Applicant has shown that pharmacologically attainableconcentrations of NaPA can promote HbF biosynthesis in the humanleukemic cells, and can cause superinduction when combined with theother chemotherapeutic agents of interest, 5AzaC and HU.

Cell Culture and reagents

The human leukemia K562 cells were maintained in RPMI 1640 mediumsupplemented with 10% heat-inactivated fetal calf serum (Gibco), 50 U/mlpenicillin, 50 μg/ml streptomycin, and 2 mM L-glutamine unless otherwiseindicated. The suspension cultures were kept in exponential growth phaseby diluting every 3-5 days with fresh medium, and cell viability wasdetermined by trypan blue exclusion. Phenylacetic acid,4-hydroxyphenylacetic acid, 3,4-dihydroxyphenylacetic acid,2,5-dihydroxyphenylacetic acid (Sigma, St. Louis, Mo.) and PAG (a giftfrom L. Trombetta, Houston, Tex.) were dissolved in distilled water, andbrought to pH 7.0 by the addition of NaOH, DON, adivicin, 5AzadC, 5AzaC,and HU (Sigma) were also dissolved in distilled water. All drug stocksolutions were stored in aliquots at -20° C. until used.

Determination of Hemoglobin Production

K562 cells were seeded at 1×10⁵ cells/ml and treated with the drugs forfour to seven days prior to assay. Qualitative estimation of hemoglobinproduction was determined by benzidine staining of intact cells insuspension. The hemoglobin concentration within cells was determined bythe protein absorption at 414 nm. Briefly, 1×10⁷ cells were lysed in 1ml of lysing buffer (0.12% Tris pH 7.4, 0.8% NaCl, 0.03% Mg-acetate, and0.5% Np-40), vortexed and incubated on ice for 15 minutes. The lysateswere then centrifuged for 15 minutes at 1500 rpm at 4° C, and theabsorption of the supernatant monitored between 350 nm and 650 nm usingBeckman Du-7 scanning spectrophotometer. The hemoglobin was quantitatedusing the relationship of 1.0 optical density (OD) at 414 nmcorresponding to 0.13 mg/ml hemoglobin as described before.

Northern Blot Analysis and DNA probes

Cytoplasmic RNA was prepared from cultures at logarithmic phase ofgrowth and separated on 1% agarose-formaldehyde gels. Gelelectrophoresis, transfer of RNA onto nytran membranes (Schleicher &Schuell), hybridization with radiolabeled DNA probes, andautoradiography (Kodak X-ray film XAR5) were according to establishedprocedures. The probe for gamma globin was a 0.6 Kb EcoRI/HindIIIfragment of the human gamma globin gene. Probes were labeled with ³²P!dCTP (New England Nuclear, Boston, Mass.) using random primed DNAlabeling kit (Boehringer Mannheim, West Germany).

Analysis of HbF Protein Synthesis

Newly synthesized proteins were labeled with ³⁵ S-methionine and the HbFimmunoprecipitated and analyzed as previously described. Briefly, cells(1×10⁶ per point in 1 ml) were first subjected to 1 hr starvation inmethionine-free medium, then incubated in the presence of 100 uCi/ml of³⁵ S-methionine for 2 hrs. The labeled cells were harvested, washed andlysed in a lysing buffer containing 10 mM phosphate buffer pH 7.4, 1%Triton×100, 0.1% SDS, 0.5% deoxycholate, 100 mM NaCl, 0>1% NaN3, 2 mMPMSF, and 10 μg/ml lenpeptin. 1×10⁷ cpm of TCA precipitable count ofcytoextract was incubated with rabbit anti-human HbF (Pharmacia) andprotein A Sepharose at 4° C., and the immunoprecipitates were separatedby electrophoresis on 12% SDS-polyacrylamide gels.

The Effect of NaPA and Analogues on Cell Growth and Differentiation

Treatment of the K562 cultures with NaPA resulted in dose dependentinhibition of cell proliferation, with 1.4 mg/ml causing 50% reductionin cell number after four days of treatment (FIG. 17). No toxicity wasobserved with doses as high as 2.0 mg/ml. In addition to the cytostaticeffect, NaPA also induced erythroid differentiation, as indicated by anincrease in the number of benzidine-positive cells (FIG. 17) andconfirmed by quantitative analysis of hemoglobin production (Table 16).Similar treatment with PAG, which is the glutamine conjugated form ofNaPA, had no significant effect on either cell proliferation orhemoglobin accumulation, suggesting that the changes associated withNaPA treatment are specific and not due to alterations in cultureconditions.

The effect of NaPA on cell growth and differentiation could be mimickedby the use of 4-hydroxyphenylacetate (Table 16). This was in markedcontrast to the analogues 3,4-dihydroxyphenylacetate and2,5-dihydroxyphenylacetate, which were highly toxic to the cells (LD50of 60 and 100 μg/ml, respectively), and did not induce differentiation.

Regulation of Fetal Hemoglobin Production by NaPA

K562 cells normally express low but detectable levels of HbF. Proteinanalysis employing anti-HbF antibodies revealed significantly increasedamounts of HbF in cells treated with NaPA compared to untreatedcontrols; this was associated with elevated steady-state levels of thefetal gamma globin mRNA. The effect of NaPA on HbF production was timeand dose dependent, and apparently reversible upon cessation oftreatment.

Glutamine Starvation and HbF Production

NaPA treatment of humans can lead to depletion of circulating glutaminedue to conjugation to glutamine and formation of PAG, an enzymaticreaction known to take place in the liver and kidney. The in vivoreduction in plasma glutamine was mimicked in vitro by culturing theK562 cells in the presence of lowered glutamine concentrations. Resultspresented in Table 17 show, in agreement with previous reports, thatglutamine starvation alone can affect the growth rate as well as HbFproduction in the K562 cells. Addition of NaPA to the glutamine-depletedgrowth medium further augmented the cytostatic and differentiatingeffects observed. Therefore, the effect of NaPA on erythroiddifferentiation and HbF production in humans may be even more dramaticthan that observed with the in vitro model, due to depletion ofcirculating glutamine and a direct effect on the erythroid progenitorcells.

Potentiation by NaPA of Erythroid Differentiation induced by OtherChemotherapeutic Drugs

There is considerable interest in the use of 5AzaC, 5AzadC and HU fortreatment of sickle cell anemia and β-thalassemia; however, the clinicaluse of these drugs is often limited by unacceptable toxicities.Combination treatments with nontoxic differentiating agents like NaPAcould enhance hemoglobin production while minimizing the adverseeffects. Therefore the efficacy of various combinations of NaPA with theother drugs of clinical interest was tested. Results, summarized inTable 18, show that addition of NaPA 800 μg/ml, to low doses of 5AzadCor HU act synergistically to further augment HbF production with notoxic effect to cells. The concentration of HU used in these experimentsis comparable to the plasma HU levels measured in sickle cell anemiapatients following an oral administration of 25 mg/kg (Goldberg et al.New England J Med 323:366-372 (1990)!. As to NaPA, pharmacokineticsstudies in children with urea cycle disorders indicate that plasmalevels of approximately 800 μg/ml can be obtained by infusion with300-500 mg/kg/day, a treatment well tolerated even by newborns.

Discussion

Chemotherapeutic agents selected for their low cytotoxic/mutagenicpotential can be used for induction of fetal hemoglobin in patients withcongenital severe anemias such as sickle cell and β-thalassemia. Drugtoxicity is an important consideration in view of overall healthcondition and the variable life-span of patients with these nonmalignantblood disorders. Unfortunately, recombinant human erythropoietin, whichhas proved to be both nontoxic and effective therapy for anemiasassociated with chronic renal disease, is apparently ineffective in thetreatment of sickle cell anemia. The application of other active drugssuch as 5AzadC, HU, vinblastine and ara-C has been hindered by concernsregarding their carcinogenic effects. HU is also difficult to usebecause of the narrow margin between toxicity and the desired effect onincreased HbF production Dover, et al., Blood 67:735-738 (1986)!. Incontrast, NaPA, shown here to affect HbF production, is so welltolerated by humans that treatment can be initiated just a few hoursafter birth.

Using an in vitro model involving human leukemic K562 cells, it is shownthat NaPA can promote the maturation of early erythroid progenitor cellsthat have an active HbF program. Addition of NaPA to other therapeuticagents currently in clinical use, i.e., 5AzaC, 5AzadC, or HU resulted insuperinduction of HbF synthesis. 5AzaC has been shown to be less toxicand more effective than HU in stimulating HbF production. Moreover,5AzaC, unlike HU, is effective in treatment of both sickle cell anemiaand β-thalassemia. Such data are consistent with the interpretation that5AzaC acts by both perturbation of erythropoiesis and by its effect onDNA methylation. However, while hypomethylation can lead to geneactivation and cell differentiation, it can also promote oncogenesis andthe evolution of cells with metastatic capabilities. Results obtainedwith the K562 erythroid progenitor cells indicate that the therapeuticeffects of NaPA compare favorably with those of 5AzadC, yet NaPA (unlikethe cytosine analog) did not cause tumor progression. Moreover, NaPA wasshown to prevent tumor progression induced by 5AzadC.

The data show that NaPA, used alone or in combination with other drugs,is of value in treatment of leukemias and β-chain hemoglobinopathies. Inaddition to promoting the production of red blood cells expressing HbFthrough nontoxic mechanisms, NaPA may also minimize the adverse effectsof other antitumor drugs currently in clinical use.

                  TABLE 16    ______________________________________    HbF Accumulation in Treated K562 Cells    Benzidine Positive               Cells           HbF production    Treatment            fold              fold    (mg/ml)    (%)       increase  (pg/cell)                                           increase    ______________________________________    None       2.2 ± 0.8                         1         0.35 ± 0.06                                           1    NaPA    0.4        2.7 ± 0.2                         1.2       0.49 ± 0.02                                           1.4    0.8        7.0 ± 0.3                         3.2       1.15 ± 0.20                                           3.3    1.6        14.6 ± 0.2                         6.6       2.40 ± 0.16                                           6.8    4HP 1.6    14.2 ± 0.5                         6.45      ND    PAG 2.6    2.1 ± 0.5                         0.95      0.37 ± 0.03                                           1.06    ______________________________________

                  TABLE 17    ______________________________________    Glutamine Starvation and HbF Production             HbF (g/cell)               Gln starvation    Gln (mM)   alone      plus NaPA (0.8 mg/ml)    ______________________________________    2.0        0.39 ± 0.04                           1.0 ± 0.06    0.5        0.56 ± 0.01                          1.15 ± 0.01    0.2.sup.a  1.17 ± 0.12                          1.75 ± 0.22    0.1.sup.a  1.86 ± 0.40                          2.22 ± 0.20    ______________________________________     .sup.a The concentration of NaPA used in this study (0.8 mg/ml) is     pharmacologically attainable without toxicity. In children such a     treatment is expected to cause a drop in circulating glutamine plasma     levels to 0.1-0.2 mM. The results presented above indicate that under suc     conditions HbF production increases 4.5-5.7 fold compared to controls. We     propose therefore that the effect of NaPA in children might be more     dramatic than that seen under routine culture conditions (i.e., cell     growth in medium with 2 mM Gln).

                  TABLE 18    ______________________________________    Potentiation by NaPA of HU's Therapeutic Effect    Treatment            HbF (pg/cell)    ______________________________________    None                 0.39 ± 0.04    NaPA (0.8 mg/ml)     1.64 ± 0.07    HU (50 uM)           1.00 ± 0.03    HU (50 uM) + NaPA    5.91 ± 0.6.sup.b    HU (100 uM)          2.12 ± 0.04    HU (100 uM) + NaPA   6.71 ± 0.05.sup.b    ______________________________________     .sup.a To mimic the effect of NaPA in vivo, treatments involving NaPA wer     performed in medium supplemented with 0.2 mM Gln (see explanation to TABL     17). Control untreated cells and those treated with HU or 5AzadC alone     were maintained in growth medium with 2 mM Gln.     .sup.b The results indicate that NaPA and HU act synergistically to induc     HbF Production int he erythroid progenitor cells     Note:     Similar results have been obtained for the combination NaPA 0.8 mg/ml and     5AzadC 0.3 uM.

Example 19

HbF induction in nonmalignant and malignant cells

General ability of NaPA and its derivatives to induce production of HbF.The ability of oral administration of sodium 4-phenylbutyrate toincrease fetal hemoglobin production was assayed. To do so, thepercentage of red cells containing fetal hemoglobin (F cells) wasmeasured by flow-cytometric single-cell immunofluorescent assays in 15patients (7 females and 8 males) with hereditary urea-cycle disorderswho had received sodium 4-phenylbutyrate therapy for 5 to 65 months. Indetermining the differences in low levels of fetal hemoglobin in personswithout anemia, the measurement of the percentage of F cells is moreprecise than conventional measurements of fetal hemoglobin as apercentage of total hemoglobin. The mean percentage of F cells wassignificantly higher in the patients than in normal subjects:

    ______________________________________                          Dose of    Patient     Age       Phenylbutyrate                                     F Cells*    No.         yr        g/kg/day   %    ______________________________________     1          29        0.30       9.4     2          11        0.67       20.4     3          6         0.62       0.5     4          5         0.48       6.5     5          2         0.58       22.7     6          13        0.46       7.7     7          2         0.38       11.8     8          11        0.41       1.9     9          6         0.27       1.9    10          5         0.62       2.3    11          6         0.65       21.1    12          21        0.29       1.7    13          3         0.47       7.6    14          6         0.64       40.5    15          2         0.63       29.7    Patients,             --         12.4 ± 3.1**    mean ± SE    Normal subjects,      --         3.1 ± 0.2    mean ± SE    ______________________________________     *F cells were measured with a flowcytometric technique that counts the     percentage of F cells in a total of 10,000 red cells. The difference     between repeated measurements was less than 10 percent.     **P = 0.005 by the KolmogorovSmirnov twosample test for the comparison of     the Fcell values in the 15 patients with ureacycle disorders and the     values in 293 normal adults. The percentage of F cells reaches the range     of values found in normal adults at about two years of age.

Example 20

In vitro study of sickle cell and beta-thalassemia responses toNaPA/NaPB

An in vitro study was conducted on cells derived from patients withhomozygous sickle cell disease or β-thalassemia who had been admitted tothe Clinical Center of the National Institutes of Health (NIH) forroutine evaluation, or normal blood donors from the Department ofTransfusion Medicine (NIH). Approximately 20-25 ml of blood was obtainedfor erythroid cell cultures. Diagnosis of SS or B-thal was made on thebasis of: (1) hemoglobin electrophoresis on alkaline cellulose acetateand on acid citrate sugar; (2) peripheral blood examination; andoccasionally (3) DNA and RNA analysis of bone marrow aspirates. Whenpossible, diagnosis was confirmed by family studies. Routine hematologicprofiles were performed on a Coulter Model S.

Peripheral blood mononuclear cells were isolated by centrifugation on agradient of Ficoll-Hypaque and cultured for 7 days (phase I) inalpha-minimal essential medium supplemented with 10% fetal calf serum(FCS) (both from GIBCO, Grand Island, N.Y.), 1 μg/ml cyclosporin A(Sandoz, Basel, Switzerland) and 10% conditioned medium collected frombladder carcinoma 5637 cultures (Myers C. D., Katz F. E., Joshi G.,Millar J. L.: A cell line secreting stimulating factors for CFU-GEMMculture. Blood 64:152, 1984). In phase II, the non-adherent cells wererecultured in alpha-medium supplemented with 30% FCS, 1% deionizedbovine serum albumin, 1×10⁵ M 2-mercaptoethanol, 1.5 mM glutamine(unless otherwise indicated), 1×10⁶ M dexamethasone, and 1 U/ml humanrecombinant Epo (Ortho Pharmaceutical Co., Raritan, N.J.). Thesecultures yielded up to 10⁶ erythroid cells per milliliter of blood. Cellviability was determined by Trypan Blue exclusion. Phenylacetic acid,4-phenylbutyric acid, p-hydroxyphenylcetic acid, p-chlorophenylaceticacid, and butyric acid (Sigma, St. Louis, Mo.) were dissolved indistilled water and brought to pH 7.0 by the addition of NaOH.5-Azacytidine and hydroxyurease was obtained from Sigma, and PAG wasobtained from S. Brusilow (Johns Hopkins, Baltimore, Md.).

Differentiation was assessed morphologically by preparing cytocentrifugeslides stained with alkaline benzidine and Giemsa. The number ofHb-containing cells was determined using the benzidine-HCl procedure(Orkin S. H., Harosi F. L., Leder P.: Differentiation of erythroleukemiccells and their somatic hybrids. Proc Natl. Acad. Sci USA 72:98, 1975).Hbs were characterized and quantitated by cation exchange highperformance liquid chromatography (HPLC) of cell lysates as previouslydescribed (Huisman T. H.: Separation of hemoglobins and hemoglobinchains by high performance liquid chromatography. J Chromatography418:277, 1987). Total Hb in lysates prepared from a known number ofHb-containing (benzidine-positive) cells was measured using either thetetramethylbenzidine procedure (Sigma kit, Catalog No. 527) or by cationexchange HPLC (measuring total area under chromatogram). Standard Hbsolutions (Isolab, Inc., Akron, Ohio) were used for reference. Meancellular Hb (MCH) was calculated by dividing the total Hb content of thelysate by the number of benzidine-positive cells.

Cytoplasmic RNA was separated on 1% agarose-formaldehyde gels. RNAisolation, gel electrophoresis, transfer onto Nytran membranes(Schleicher & Schuell, Inc., Keene, N.H.), hybridization withradiolabeled DNA probes, and autoradiography (Kodak X-ray film XAR5)were described Samid D., Yeh A., Presanna P.: Induction of erythroiddifferentiation and fetal hemoglobin production in human leukemic cellstreated with phenylacetate. Blood, 80:1576, 1992!. The human globin cDNAprobes included JW101 (alpha), JW102 (beta), and a 0.6 kb EcoRI/HindIIIfragment of the 3' end of human G-gamma-globin gene. Probes were labeledwith ³² P!dCTP (New England Nuclear, Boston, Mass.) using a randomprimed DNA labeling kit (Boehringer, Mannheim, Germany).

Results

Addition of NaPA or NaPB to phase II erythroid cultures resulted inreduced cell proliferation with no apparent change in cell viability.Cytostatis was associated with a decline in total Hb produced perculture; however, both Hb content per cell (MCH) and the proportion ofHbF (%HbF) increased upon treatment (FIG. 18). The extent of changesobserved was dose- and time-dependent: the earlier the drugs were addedduring the second phase of growth, the higher was the increase in % HbF,however, cell yields were proportionately decreased. For example,addition of 5 mM NaPA to normal precursors on day 2 caused approximately90% decrease in cell number along with a 12-fold increase in % HbF, adetermined on day 13. When treatment was initiated on day 67, cellnumber decreased on by 60% compared to controls, and % HbF increased3.3-fold. In order to obtain sufficient cells for further analysis,subsequent experiments involved the addition of drugs on days 6-7, andcells were harvested on day 13. Under these conditions, results werereproduced in cultures derived from 6 normal donors as well as 4patients with sickle cell anemia and 4 patients with β-thal. NaPA (5 mM)and NaPB (2.5 mM) caused a significant increase in both MCH(38-100%) andthe proportion of HbF produced. In the case of homozygous SS patients, %HbF was elevated 2.0-4.1 fold (mean 3.0) by 4 mM NaPA, and 3.2-5.6 fold(mean 4.0) by 2.5 mM NaPB. The latter was associated with a 12±3%decrease in HbS levels, with no change in HbA₂ (FIG. 19).

As in K562 cells, increased HbF production by NaPA or NaPB in primarycultures of normal or SS cells appears to be due to pre-translationalregulation of gamma-globin expression. Northern blot analysis showeddose-dependent increase (up to 5 fold) in the steady-state levels ofgamma globin mRNA, accompanied by a slight decrease (less than two fold)in the amounts of beta globin transcripts. There was no change in alphaglobin expression.

PAG, the end-metabolite of both NaPB and NaPA, is formed byphenylacetate conjugation to glutamine with subsequent excretion in theurine. PAG was found to be inactive on erythroid proliferation and HbFaccumulation. Glutamine starvation of the non-malignant erythroid cellshad no effect on either cell growth or HbF production, nor did itenhance the efficacy of NaPA.

The effect of NaPA with other drugs was also assayed. When used alone incultures derived from normal donors (HbF base levels of 0.8-2.0%), NaPA(5 mM) and hydroxyurease (0.05 mM) increase % HbF by 3.5 and 2.0-fold,respectively; the combination of the two resulted in a 4.7-fold increasein HbF. NaPA also augmented HbF stimulation by butyrate (0.5 mM) (from3.1 to 7.15-fold), and of 5-Azacytidine (2 μM) (from 2.5 to 6.6-fold).These results indicate that NaPA when added to suboptimal, non-toxicdoses of other drugs, can potentiate HbF production with significantcytostasis and no significant change in cell viability.

As exemplified below in Table 20, combination treatment comprisingadministration of NaPA (or a pharmaceutically acceptable derivative ofphenylacetic acid) simultaneously with hemin, a known stimulator of HbFproduction, synergistically increases the induction of erythroiddifferentiation, as indicated by the increase in the number of benzidinepositive cells, and HbF production. In K562 cells, the range of increasein the production of HbF with this combination treatment varied from 1.5to 5 times that produced by treatment with 10 mM PA alone. Further,treatment with NaPB in combination with hemin also resulted in classicalsynergism. Similar results were also obtained with PB in non-malignanterythroid progenator primary cells. In all cases, treatment with bothdrugs was maintained for 4-6 days prior to measurement of HbF.

                  TABLE 20    ______________________________________    STIMULATION OF HbF BY NaPA IN COMBINATION WITH    HEMIN - K562 MODEL                % Benzidine    R.sub.x     pos.        Hb pg/cell                                     Viability    ______________________________________    CONTROL     >0.01       0.26     97    NaPA (10 mM)                1.6-3.1     0.91     96    NaPA (10 mM) + H                25.4-32.6   4.03     92    NaPA (5 mM) + H                12.6        2.34     99    NaPA (2.5 mM) + H                8.1         1.95     94    HEMIN (20 μM)                2.9         1.04     98    CONTROL     2.1         0.65     97    NaPA (2.5 mM)                2.6         0.91     97    NaPA (5 mM) 7.7         1.04     97    NaPA (10 mM)                14.3        nd       96    HEMIN (20 μM)                13.8        2.34     nd    NaPA (5 mM) + H                42.3        5.2      97    ______________________________________

Section D: Use of Phenylacetic Acid and its Derivatives in Wound Healing

Growth factors, including TGF-α, play a critical role in wound healingand repair processes. Wound healing is a localized process that involvesinflammation, wound cell migration and mitosis, neovascularization, andregeneration of the extracellular matrix. Recent data suggest the actionof wound cells may be regulated by local production of peptide growthfactors which influence wound cells through autocrine and paracrinemechanisms (Schultz et al., J. Cell Biochem. 45(4):346 (1991); Schultzet al., Acta Ophthalmol. Suppl.(Copenh), 202:60 (1992)). Two peptidegrowth factors which may play important roles in normal wound healing intissues such as skin, cornea, and the gastrointestinal tract are thestructurally related epidermal growth factor (EGF) and TGF-α, whosereceptors are expressed by many types of cells including skinkeratinocytes, fibroblasts, vascular endothelial cells, and epithelialcells of the gastrointestinal tract. EGF or TGF-α is synthesized byseveral cells involved in wound healing, including platelets,keratinocytes, activated macrophages and corneal epithelial cells.Healing of a variety of wounds; in animals and patients, such asepidermal regeneration of partial thickness burns, dermatome wounds,gastroduodenal ulcers and epithelial injuries to the ocular surface, isenhanced by exogenous treatment with EGF or TGF-α. TGF-α, which is apotent inducer of lysyl oxidase mRNA levels in cultures of human scleralfibroblasts, may be primarily responsible for inducing synthesis ofextracellular matrix components after an injury. Furthermore, TGF-α isknown to promote angiogenesis.

The lack of adequate stimulation of growth factors contributes to thenonhealing conditions of many chronic wounds. Poorly healing conditionscould markedly benefit from either addition of exogenous TGF-α orstimulation of effector cells to produce TGF-α and related growthfactors. It has now been discovered that PA and PB (or apharmaceutically acceptable derivative) are capable of stimulatingproduction of TGF-α in cells of melanocytic origin; astrocytic lineage(glioblastoma cells); and several normal human epithelial cell types,including keratinocytes (FIG. 20), which are involved in wound healing.Further, treatment with PA and PB enhances collagen-a type 1 expression.Induction of TGF-α mRNA expression upon treatment with NaPA and NaPB inhuman melanoma cells; was observed expression of TGF-α was confirmedfollowing protein analysis. FIG. 20 shows the increased production ofthe TGF-α protein in human keratinocytes upon exposure to NaPA and NaPB.This increased production of TGF-α is maintained for a few days afterwhich the levels return to approximately pretreatment levels. Asdiscussed below and in FIG. 21, further support for the use thesecompounds in treating wounds may be found in the enhanced expression ofICAM-1, which is a cellular adhesion molecule/surface antigen, followingtreatment with NaPB.

Thus, the instant invention provides a method for stimulating theproduction of TGF-α in cells. Further, wound healing in a human oranimal can be enhanced by treatment with a therapeutic amount ofphenylacetic acid or a derivative of phenylacetic acid such as NaPA orNaPB, which stimulates the in-situ production of TGF-α. For instance,surface wounds can be treated by topically applying PA, PB or aderivative of either PA or PB to the skin surface, such as in a creamformulation. Likewise, ocular injuries can be treated by application ofa PA or PB (or PA/PB derivative) formulation, such as eye drops, to thecornea. Similarly, internal injuries, such as injuries to thegastrointestinal tract, can be treated by administration of oralformulations. Vaginal or anal injuries can also be treated, such as witha suppository containing pharmaceutically effective amounts of PAA or aderivative. The PA/PB or derivative formulations can be administeredcontinuously or, preferably, intermittently, such as one or more dosesin daily, weekly or monthly courses. For example topical administrationonce or twice a day of a composition containing from 0.1 to 10 mM PA,preferably 0.1 to 5.0 mM PA or from 0.1 to 5 mM PB, preferably 0.1 to2.5 mM PB over the course of a week adequately stimulates wound repair.From the information contained herein, dosage concentrations and amountsfor the various administration vehicles can be easily determined. Forinstance, a topical treatment, such as a cream containing PB, typicallywill contain approximately 0.5 to 3.0 mM PB or an equipotent (byequipotent it is meant that dosage may be varied among the differentphenylacetic acid derivatives so as to achieve the equivalent effect onthe subject) dose of a phenylacetic acid derivative. For instance, andwithout limitation, approximately one-half as much PB in a dose isneeded to equal the potency of a similarly indicated PA dose.

Section E: Use of Phenylacetic Acid or its Derivatives in Treatment ofDiseases Associated with Interleukin-6

Interleukin-6 (IL-6), which can be produced by monocytes andkeratinocytes upon stimulation, is a pleiotropic cytokine that plays acentral role in defense mechanisms, including the immune response, acutephase reaction and hematopoiesis. Activation of mature B cells can betriggered by antigen in the fluid phase. When antigen binds to cellmembrane IgM in the presence of IL-1 and IL-6, mature virgin B cellsdifferentiate and switch isotypes to IgG, IgA or IgE. Abnormalexpression of the IL-6 gene has been suggested to be involved in thepathogenesis and/or symptoms of a variety of diseases, including (1)non-malignant disorders associated with abnormal differentiationprograms, autoimmunity and inflammatory processes, e.g., rheumatoidarthritis, Castleman's disease, mesangial proliferation,glomerulonephritis, uveitis, sepsis, autoimmune diseases such as lupus,inflammatory bowel, type I diabetes, vasculitis, and several skindisorders of cell differentiation such as psoriasis and hyperkeratosis;(2) viral diseases such as AIDS and associated neoplasms, e.g., Kaposi'sSarcoma and lymphomas; and (3) other neoplasms, e.g., multiple myeloma,renal carcinoma, Lennert's T-cell lymphoma and plasma cell neoplasms.For instance, significantly increased IL-6 mRNA levels in lesionalpsoriatic tissue relative to normal tissue and elevated amounts of IL-6in sera and peripheral blood mononuclear cells of psoriatics compared tosamples from atopics or healthy controls have been found (Elder et al.,Arch. Dermatol. Res., 284(6):324 (1992); Neuner et al., J.

Invest. Dermatol., 97(1):27 (1991)).

It has now been discovered that phenylacetic acid or a derivative ofphenylacetic acid, such as NaPA or NaPB, can inhibit the expression ofIL-6. For instance, PA inhibits IL-1-induced IL-6 expression in coloncarcinoma cells. This reduction in RNA is confirmed by reduction in IL-6protein. Thus, PA, PB and their derivatives can be used in the treatmentof diseases involved with the abnormal overexpression of IL-6.

For instance, treatment twice daily by topical application of either 2mM NaPB in a mineral oil-based cream or 2mM napthylacetate and VitaminB₁ in a mineral oil-based cream directly onto the patient's psoriaticlesions resulted in disappearance of the lesions within a week. Similartreatment of a patient with a severe case of psoriasis resulted in thepsoriatic lesions resolving in approximately 1-3 weeks. Obviously, themode of administration and amount of drug can vary depending upon theIL-6-related disease being treated in order to target the drug to thecells in which reduction of IL-6 expression is desired. For example,injection of a 0.1 mM-5mM PB solution or an equipotent solutioncontaining a pharmaceutically acceptable phenylacetic acid derivativeinto the joint region may be appropriate for treatment for rheumatoidarthritis whereas other diseases may be more appropriately treated bytopical, intravenous or oral delivery. Treatment can be by eithercontinuous or discontinuous treatment, but cessation of the drug,particularly PB, may be accomplished by ramping down the dosage amountsto prevent an overreaction to the cessation of treatment with the drug.Additionally, diseases involving the abnormal overexpression of IL-6 canbe treated by administration of an effective amount of phenylacetic acidor a phenylacetic acid derivative, particularly PA or PB, in combinationwith an effective amount of an anti-inflammatory agent, includingvarious vitamins such as vitamin B₁, non-steroidal inflammatory agentsand steroidal anti-inflammatory agents. The anti-inflammatory agent canbe combined with the phenylacetic acid derivative(s) of this inventionin the same dosage form or administered separately by the same ordifferent route as the derivative. An effective amount of theanti-inflammatory agent refers to amounts currently in clinical use forthe specific disease state or less.

Section F: Use of Phenylacetic Acid or its Derivatives in The Treatmentof Aids-Associated CNS Dysfunction

Hallmarks of central nervous system (CNS) disease in AIDS patients areheadaches, fever, subtle cognitive changes, abnormal reflexes andataxia. Dementia and severe sensory and motor dysfunction characterizemore severe disease. Autoimmune-like peripheral neuropathies,cerebrovascular disease and brain tumors are also observed. In AIDSdementia, macrophages and microglial cells of the CNS are thepredominant cell types infected and producing HIV-1. However, it hasbeen proposed that, rather than direct infection by HIV-1, the CNSdisease symptoms are mediated through secretion of viral proteins orviral induction of cytokines that bind to glial cells and neurons, suchas IL-1, TNF-α and IL-6 (Merrill et al., FASEB J., 5(10):1291(1991)).TGF-β is a growth factor which is released by many cell types. Amongother effects, TGF-β is highly chemotactic for macrophages andfibroblasts and stimulates the release of TNF-α, TGF-α and, indirectly,a variety of other modulators from macrophages which have beenimplicated in the initiation of the CNS symptoms of AIDS.

It has now been discovered that phenylacetic acid or a derivative ofphenylacetic acid, such as NaPA or NaPB, can inhibit the production ofTGF-β2. Because TGF-β2 is an immunosuppresive factor, this inhibitionresults in a general improvement of the patient's immune system. Gene,gene expression of TGF-β2 in glioblastoma cells was inhibited by both PAand PB. This reduction in RNA leads to reduced TGF-β2 protein synthesis.Thus, PA, PB or their derivatives can be used to inhibit the productionof TGF-p2 in cells, particularly to control or alleviate the CNSsymptoms resulting from HIV infection. As discussed above, thistreatment also inhibits the production of IL-6, further allowing foralleviation of the CNS symptoms. Amounts of drug and/or regimens ofadministration effective for inhibiting TGF-β2 correspond to thoseappropriate for treatment or prevention of cancer as given herein, suchas in SECTION C.

Section G: Use Of Phenylacetic Acid And Its Derivatives To EnhanceImmunosurveillance

Immunosurveillance in an animal such as a human can be enhanced bytreatment with PA, PB or their derivatives. Tumor cells are thought toescape attack by the immune system by at least two means. First, manytumors secrete immune suppressive factors that directly reduce immuneactivity. Additionally, some tumor cells do not express, or have reducedexpression of, appropriate surface antigens that allow the immune systemto identify outlaw cells. However, the compositions of the instantinvention can activate otherwise dormant genes such as fetal globin,perhaps by DNA hypomethylation. Similarly, activation of cancersuppressor genes, dormant antigens and other genes, such as (1) cellularmajor histocompatibility antigens (MHC Class I and II) or other surfaceantigens, such as ICAM-1; (2) tumor antigens such as MAGE-1; and (3)viral latent proteins such as EBV's latent membrane protein (which isimplicated in numerous diseases such as T-cell neoplasms, Burkitt'slymphoma nasopharyngeal carcinoma, and Hodgkin's disease), maycontribute to enhanced immunosurveillance. Thus, neoplastic cells can betreated with PA, PB or their derivatives to provide for expression ofcell surface antigens that increase the effectiveness of the immunesystem by allowing for adequate identification and clearance of thetumor cells by the immune system. Activation of latent viral proteinscould also induce a lytic cycle leading to death of the infected cell.

Evidence that the instant phenylacetic acid or phenylacetic acidderivative compositions can activate dormant genes and enhanceexpression of surface antigens is given by FIG. 21, which shows enhancedexpression of MHC Class I, MHC Class II and the adhesion molecule ICAM-1in melanoma cells that have been treated for 10 days with 2 mM NaPB(e.g., note the shift of the population mean from approximately 50 to200 for MHC class I).

Furthermore, it has now been discovered that PB induces expression ofEBV's latent membrane protein (LMP) in Burkitt's lymphoma cells.Cytoplasmic RNA (20 μg/lane) was isolated from LandisP, RajI and P3HRIBurkitt's lymphoma cell lines, which had been treated with 2 mM PB forfour days, and subjected to Northern blot analysis with a specific LMPprobe. In all three cell lines, a positive reaction was observedcompared to controls (untreated cells), indicating that PB induces theexpression of EBV's latent membrane protein. In Burkitt's lymphoma cellsboth PA and PB cause additional molecular and cellular changes,including cytostasis, decline in myc expression and enhancement ofHLA+1.

Because these surface antigens enhance tumor immunogenicity in vivo,treatment of the animal (human) with PA, PB or their derivatives canenhance the effectiveness of the immune system of the individual. Dosesof approximately 0.5-3.0 mM PB or equipotent doses of pharmaceuticallyacceptable phenylacetic acid derivatives may be useful. This treatmentcan also be combined with conventional immunotherapy treatments and/orantigen targeted, antibody-mediated chemotherapy. While treatmentusually is accomplished by a protocol which allows for substantiallycontinuous treatment, discontinuous or pulsed treatment protocols arealso effective, especially for cells capable of terminallydifferentiating upon treatment with PAA or a PAA derivative. Forinstance, treatment of the melanoma cells given in FIG. 21 for 10 dayswas sufficient to allow continued enhanced expression of the surfaceantigens past this 10 day period.

Example 21

NaPA and NaPB Effects on Burkitt's Lymphoma

The Epstein Barr Virus (EBV) infected cell line is growth inhibited byNaPB more than by NaPA. This may be due to a reduction c-myc expressionafter about four days of treatment. Unfortunately, there is at thepresent time, no known measure of differentiation in this cell line.However, treatment with NaPA causes substantial morphological changesand clumping (perhaps due to cell-cell or cell-substrate interactionalterations). The increase in proportion of cells which bind to theextracellular matrix may be due to an increase in ICAM 1. The cellproduces more HLA class I antigens and more latent membrane proteinwhich thereby enhances the visibility of the cell to the immune system.

Section H: Method of Monitoring the Dosage Level of Phenylacetic Acid orits Derivatives in a Patient and/or the Patient Response to these Drugs

As discussed above, administration of phenylacetic acid or a derivativeof phenylacetic acid such as NaPA or NaPB to an animal (human) inamounts and over treatment courses as described herein induce a varietyof molecular changes. These molecular traits can be used as biomarkersto either (1) monitor the dosage level of the drug or itsbioavailability in the animal and/or (2) serve as a biomarker of thepatient response to the drug. For instance, as described above,administration of an effective amount of NaPA or NaPB (or theirderivatives) results in a variety of molecular effects, including a)increased levels of fetal hemoglobin in erythrocytes; b) increasedproduction of TGF-α in various cells such as those of melanocyticorigin, astrocytic lineage or epithelial cell types; c) inhibition ofthe production of IL-6; and d) inhibition of the production of TGF-β2.Thus, absolute or relative (before/after treatment) concentrations of aparticular biomarker can be determined in an appropriate cell populationof the individual to allow monitoring of the dosage level orbioavailability of the drug. Further, this concentration can becorrelated or compared with patient responses to develop a patientresponse scale for a desired treatment goal based upon that biomarker.For instance, the increased amount of fetal hemoglobin can be used toindicate the bioavailability of PA or PB for treatment or prevention ofa neoplastic condition as well as indicating the degree of patientresponse to the drug.

Section I: The Activation of the PPAR by Phenylacetic Acids and itsDerivatives

Peroxisomes are cellular organelles that contain enzymes which controlthe redox potential of the cell by metabolizing a variety of substratessuch as hydrogen peroxide. Recent advances in this area reveal thatperoxisomes can be proliferated through activation of a nuclear receptorwhich regulates the transcription of specific genes (Gibson, Toxicol.Lett., 68(1-2):193(1993).). This nuclear receptor has been named theperoxisome proliferator-activated receptor (PPAR) and belongs to thesteroid nuclear receptors family that have a major effect on geneexpression and cell biology. Binding by peroxisome proliferators such asclofibrate, herbicides, and leukotriene antagonists with PPAR activatesthe nuclear receptor, which acts as a transcriptional factor, and cancause differentiation, cell growth and proliferation of peroxisomes.Although these agents are thought to play a role in hyperplasia andcarcinogenesis as well as altering the enzymatic capability of animalcells, such as rodent cells, these agents appear to have minimalnegative effects in human cells, as exemplified by the safety of drugssuch as clofibrate (Green, Biochem. Pharm. 43(3):393(1992)).

Peroxisome proliferators typically contain a carboxylic functionalgroup. Therefore, PA, PB and various phenylacetic acid derivatives weretested for their ability to activate the PPAR and compared with knownperoxisomal proliferators. As shown in FIG. 22, Clofibrate, a knownactivator of peroxisomal proliferation, caused a 4- to 5-fold increasein activation as measured by increased production of the responseelement for acyl-CoA oxidase, which is the rate limiting enzyme inbeta-oxidation and is contained in peroxisomes (Dreyer et al., Biol.Cell, 77(1):67(1993)). PA and PB caused mild activation (double baselineactivity), naphthyl acetate was relatively more active (approximately2.5- to 4-fold increase) while the halogenated analogs of PB were verypotent stimulators. Interestingly, butyrate was not a significantperoxisomal proliferation activator.

The peroxisome proliferator-activated receptor has been shown to belongto the same family of nuclear receptors as the retinoid, thyroid andsteroid receptors and PPAR is known to interact with RXR, the receptorof 9-cis-retinoic acid (a metabolite of all-trans-retinoic acid).Because the PPAR signaling pathway converges with the 9-cis retinoidreceptor signal, it can be anticipated that retinoic acid or the likewill significantly enhance the activity of PA or PB or otherphenylacetic acid derivatives of this invention. Indeed, enhancement ofthe induction of HL-60 cell differentiation by NaPA in combination withretinoic acid is discussed above. Additionally, this synergisticresponse has been confirmed in other tumors, such as neuroblastoma,melanoma and rhabdomyosarcoma cells.

Thus, combination therapy consisting of administration (simultaneouslyin the same dosage form or simultaneously/sequentially in separatedosage forms) of Vitamin A, Vitamin D, Vitamin C, Vitamin E, B-carotene,or other retinoids and the like with PA, PB or other phenylacetic acidderivatives is encompassed by the instant invention for any of thetreatment regimes given herein. Appropriate doses of the phenylaceticacid derivatives include approximately 0.5-10 mM PA, more preferably0.5-5mM PA, doses or equipotent doses of a pharmaceutically acceptablephenylacetic acid derivative. Between 0.1 and 1.0 μM concentrations ofthe retinoids are expected to be effective. This combination therapyenhances, for instance, the efficacy of treatment with PA, PB or otherphenylacetic acid derivatives, taken alone, for cancer, anemia and AIDStreatment, wound healing, and treatment of nonmalignant disorders ofdifferentiation.

Agents affecting cellular peroxisomes have a major impact on oxidativestress and the redox state of a cell. Thus, further evidence that PA, PBor other phenylacetic acid derivatives activate PPAR can be found by therapid increase of gamma glutamyl transpeptidase and catalase followingcellular exposure to PA or PB as shown in FIG. 23. These antioxidantenzymes, whose activities are increased when peroxisome proliferationhas been activated, were increased by 100% 24 hours after administrationof sodium phenylbutyrate. This effect was reversed by approximately 48hours and activity was maintained below control levels through 100hours. The intracellular level of glutathione followed a similarbiphasic pattern with an initial increase (20%) followed by a fall tolevels below baseline at 100 h. The rapid induction with subsequentsharp decline of these antioxidant enzymes was observed in numeroustumor types from prostatic, breast and colon adenocarcinomas,osteosarcoma, and brain tumors. Molecular analysis showed changes in therate of gene transcription of the GSH-related and antioxidant enzymes,which are consistent with activation of PPAR by PA, PB or their analogs.

Because peroxisomal enzymes are instrumental in defending againstoxidative stress, experiments were undertaken to examine the effects oftreatment with PA or PB on cells which were subjected to chemical orradiation stress. Pretreatment of glioblastomas (FIG. 24), breastcarcinoma and metastatic prostate cells with a non-toxic dose of PA orPB 72 hours prior to CO⁶⁰ γ-radiation or treatment with adriamycindemonstrated a significant dose-related increase in cell killing byeither modality. The surviving fraction of cells following drugtreatment was nearly one tenth the fraction surviving with nopretreatment, which suggests that PA, PB or other like analogs could beused to increase the efficacy of radiation therapy and chemotherapysubstantially. As such, the instant invention encompasses combinationanti-cancer therapy consisting of administration of an non-toxiceffective amount of phenylacetic acid or a pharmaceutically acceptablephenylacetic acid derivative (according to any of the dosageconcentration protocols given herein) in combination with radiationtherapy, particularly local treatment, or chemotherapy, particularlytargeted to the tumor cells. This adjuvant therapy can be administered,for instance, after approximately 24 hours, such as from 24 hours to 120hours or more, from the initiation of the administration of thederivative.

Phenyl fatty acids such a PA and its derivatives or analogs haveexhibited a wide range of activity in the treatment of glioblastomas andadenocarcinomas by inducing malignant cell differentiation at relativelynontoxic doses. Further, it has been discovered that PA and itsderivatives/analogs inhibit the post-translational processing of the rasoncogene-encoded protein which was implicated in the maintenance oftumor cell radioresistance. Thus, PA and its derivatives/analogs may beuseful and potent radiosensitizers. The lack of significant toxicity forPA/PB makes them particularly attractive radiosensitizers in comparisonto currently used chemical sensitizers.

In vitro data demonstrate that cellular exposure to PA and PB resultedin a marked increase in cellular radiosensitivity in brain, breast,prostate, and colon human tumor cells. This increased radiosensitivitywas associated with a decrease in the activity of protective antioxidantenzymes such as catalase and gamma-glutamyl transferase and aconcomitant decrease in the naturally occurring radioprotector,glutathione (GSH). Interestingly, highly radioresistant prostate tumorcells that express high levels of the ras oncogene were significantlyradiosensitized in comparison to tumor cells that express low levels ofras which were only slightly radiosensitized by PA exposure.

These results suggest a further consideration of a variety of pathogenicdisorders. Inflammatory response to tissue injury, pathogenesis ofemphysema, ischemia-associated organ injury (shock), doxorubicin-inducedcardiac injury, drug-induced hepatotoxicity, atherosclerosis, andhyperoxic lung injuries are each associated with the production ofreactive oxygen species and a change in the reductive capacity of thecell. Although long-term exposure to PA or other phenylacetic acidanalogs depletes cellular redox protection systems, short term treatmentwith PA and the like may have significant implications for treatment ofdisorders associated with increased reactive oxygen species.

Section J: Use of Phenylacetic Acid and its Derivatives in Treatment ofCancers Having a Multiple-Drug Resistant Phenotype

In treating disseminated cancers, systemic treatment with cytotoxicagents is frequently considered the most effective treatment. However, anumber of cancers exhibit the ability to resist the cytotoxic effects ofthe specific antineoplastic drug administered as well as other agents towhich the patient's system has never been exposed. In addition, somecancers appear to have multiple drug resistance even prior to the firstexposure of the patient to an antineoplastic drug. Three mechanisms havebeen proposed to explain this phenomenon: P-glycoprotein Multiple DrugResistance (MDR), MDR due to Topoisomerase Poisons and MDR due toaltered expression of drug metabolizing enzymes (Holland et al., CancerMedicine, Lea and Febiger, Philadelphia, 1993, p. 618-622).

P-glycoprotein MDR resistance appears to be mediated by the expressionof an energy-dependent pump which rapidly removes cytotoxic agents fromthe cell. High levels of p-glycoprotein are associated withamplification of the MDR gene and transcriptional activation. Increasedexpression of p-glycoprotein can also be stimulated by heat shock, heavymetals, other cytotoxic drugs and liver insults, and ionizing radiationin some cell lines from some species. The results are not sufficientlyconsistent to confirm a causal relationship but are highly suggestive.

Topoisomerases are nuclear enzymes which are responsible for transientDNA strand breaks during DNA replication, transcription andrecombination. Cytotoxic agents, such as etoposide, doxorubicin,amsacrine and others are known poisons of topoisomerase II, and causelethal DNA strand breakage by the formation of stable complexes betweenthe DNA, topoisomerase II and drug. MDR to this type of drug is thoughtto be caused by changes in the nature and amount of enzymatic activity,which is thought to prevent the formation or effect of theDNA-enzyme-drug complex.

Some cytotoxic agents are able to induce increased metabolic capabilitywhich permits rapid elimination of the toxin. Among the enzymes whichhave been implicated are glutathione S-transferase isozymes (GSTs).These enzymes are responsible for the conjugation of the electrophilicmoieties of hydrophobic drugs with glutathione, which leads todetoxification and elimination of the drug.

As discussed above, PA and other phenylacetic acid analogs have beenshown to stimulate the proliferation of peroxisomes which contain someisozymes of GST. Based on that observation, it would be expected that PAand PB would also stimulate MDR. However, as shown in FIG. 25, it hasnow been discovered that the opposite occurs. Thus, FIG. 25 shows theinhibition by PA of the growth of cells from a line of breast cancercells that exhibit the MDR phenotype. Up to 10 mM PA in cultures, growthof cells is dramatically inhibited in a dose-dependent manner.Surprisingly, PA and PB are more highly active againstadriamycin-resistant breast cancer cells than compared toadriamycin-sensitive cells. This increased sensitivity of the MDRphenotype is reproducible in other tumor models, including those thatare resistant to radiation therapy.

Thus, the instant invention provides a method of treating tumor cellpopulations in a patient that are resistant or able to survive currentconventional treatments, particularly tumors having a MDR phenotype, byadministration to the patient of non-toxic amounts of PA (such asamounts that provide up to 10 mM PA or an equipotent dose of apharmaceutically acceptable phenylacetic acid derivative) in thevicinity of the tumor or equivalently effective amounts of phenylaceticacid or a phenylacetic acid analog. PA or other analog dosage protocolssimilar to those described in relation to the potentiation ofdifferentiation in tumor cells by these phenylacetic acid-relatedcompounds; including the various combination therapies described herein,can be used to treat patients with resistant tumors such as MDR tumors.Long-term (weeks, months) or short-term (day(s)) substantiallycontinuous treatment regimens (including continuous administration orfrequent administration of separate doses) as well as pulsed regimens(days, weeks or months of substantially continuous administrationfollowed by a drug-free period) can beneficially be employed to treatpatients with MDR tumors.

Section K: Phenylacetate and its Derivatives, Correlation BetweenPotency and Lipophilicity

One potential problem that could hinder the clinical use ofphenylacetate is related to the large amounts of drug required toachieve therapeutic concentrations, i.e., over 300 mg/kg/day. Studieswere thus undertaken to develop analogs that are effective at lowerconcentrations. Studies in plants revealed that increasing thelipophilicity of a phenylacetate analogue (as measured by itsoctanol-water partition coefficient) enhanced its growth-regulatoryactivity Muir, R. M., Fujita, T., and Hansch, C. Structure-activityrelationship in the auxin activity of mono-substituted phenylaceticacids. Plant Physiol., 42: 1519-1526, 1967.!. Calculated partitioncoefficient (CLOGP) was used to correlate the predicted lipophilicitywith the measured antitumor activity of phenylacetate analogues. Forthese analogues, enhanced potency in inducing cytostasis and phenotypicreversion in cultured prostate carcinoma, glioblastoma, and melanomacells was correlated with increased drug lipophilicity.

Cell Cultures

Studies included the following humans tumor cell lines: (a)hormone-refractory prostatic carcinoma PC3, DU145, purchased from theAmerican Type Culture Collection (ATCC, Rockville, Md.); (b)glioblastoma U87, A172 (ATCC);(c) melanoma A375 and mel 1011, providedby J. Fidler (M. D. Anderson, Houston Tex. and J. Weber (NCI, BethesdaMd.), respectively. Cells were maintained in RPMI 1640 supplemented with10% heat inactivated fetal calf serum (Gibco Laboratories), antibiotics,and 2 mM L-glutamine. Diploid human foreskin FS4 fibroblasts (ATCC), andhuman umbilical vein endothelial cells (HUVC) were used for comparison.The HUVC cells, isolated from freshly obtained cords, were provided byD. Grant and H. Kleinman (NIH, Bethesda Md.).

Antitumor Agents

Sodium phenylacetate and phenylbutyrate were from Elan Pharmaceuticalcorp, Gainvesville Ga. 4-Iodophenylacetate, 4-iodophenylbutyrate and4-chlorophenylbutyrate were synthesized by the Sandmeyer procedure fromthe corresponding 4-amino-phenyl-fatty acids. The halogenated productswere extracted from the acidic reaction mixtures with diethyl etherwhich was then taken to dryness. The residue was dissolved in boilinghexane and the crystals that formed on cooling were collected by suctionfiltration. The product was recrystallized from hexane until thereported melting points were obtained. Amides of phenylacetate andphenylbutyrate were produced by heating the sodium salts with a smallexcess of thionylchloride followed by the addition of ice-coldconcentrated ammonia. The amides were purified by recrystalization fromboiling water. The identity of synthesized compounds was verified bymelting point determination and by mass spectroscopy. All commerciallyavailable derivatives were purchased from Aldrich (Milwaukee, Wis.) orSigma (St. Louis, Mo.), depending on availability. Tested compounds wereall dissolved in distilled water, brought to pH 7.0 by the addition ofNaOH as needed, and stored in aliquots at -20° C. till used.

Calculation of Relative Drug Lipophilicities

Estimation of the contribution of lipophilicity to the biologicalactivity of a molecule was based on its calculated logarithm ofoctanol-water partition coefficient (CLOGP). This was determined foreach compound using the BLOGP program of Bodor et al., (BLOGP version1.0, Center for Drug Discovery, University of Florida) assuming that thedegree of ionization is similar for all tested compounds.

Quantitation of Cell Growth and Viability

Growth rates were determined by cell enumeration with a hemocytometerfollowing detachment with trypsin-EDTA, and by an enzymatic assay using3- 4,5-dimethylthiazol-2-yl!-2,5-diphenyltertrazolium bromide (MTT).These two assays produced essentially the same results. Cell viabilitywas assessed by trypan blue exclusion.

Colony Formation in Semi-Solid Agar

For analysis of anchorage independent growth, cells were harvested withtrypsin-EDTA and resuspended at 1.0×10⁴ cells per ml in growth mediumcontaining 0.36% agar (Difco). Two ml of cell suspension were added to60 mm plates (Costrar) which were pre-coated with 4 ml of solid agar(0.9%). Tested drugs were added at different concentrations, andcolonies composed of 30 or more cells were counted after 3 weeks.

Growth on Matrigel

Cells were first treated with drugs in T.C. plastic dishes for 4-6 days,and then replated (5×10⁴ cells per well) onto 16 mm dishes (Costar,Cambridge, Mass.) coated with 250 ul of 10 mg/ml matrigel, areconstituted basement membrane (Collaborative Research). Drugs wereeither added to the dishes or omitted in order to determine thereversibility of effect. Net-like formation characteristic of invasivecells occurred within 12 hours, while invasion into the matrigel wasevident after 6-9 days.

Drug Uptake Studies

Cells were plated in 6-well T.C. dishes (Costar) at 5×10⁵ cells perdish. The growth medium was replaced after 24 hrs with 750 ul of freshmedium containing 4.5×10⁵ DPM of either ¹⁴ C-phenylacetic acid (3.4mCi/mmmol, Sigma) or ¹⁴ C-naphthylacetic acid (5.4 mCi/mmol, Sigma), andthe cultures were incubated for 10-180 minutes at 37° C. Labeling wasterminated by placing plates on ice. Cells were then washed twice with 5ml ice-cold phosphate buffer saline (PBS), detached by scraping, and theradioactivity retained by cells determined using liquid scintillation.Blank values were determined by incubating the radiolabled compounds inan empty dish.

Correlation Between Drug Lipophilicity and Growth-Inhibitory Effect ofPhenylacetate and its Analogues

The growth inhibitory effect of these compounds on prostatic carcinoma,glioblastoma, and melanoma cell lines are expressed as IC₅₀ andcorrelated with drug lipophilicity determined using the CLOGP program.As seen in Tables 21 and 22, there is a good correlation betweencytostasis and lipophilicity. In agreement with previous observationswith phenylacetate (3), the cytostatic effect was selective as higherdrug concentrations were needed to significantly affect theproliferation of normal endothelial cells and skin fibroblasts. Nocytotoxicity (i.e., decline in cell viability) occurred during 4-6 daysof continuous treatment with the tested compounds.

                  TABLE 21    ______________________________________    Phenylacetate and analogues containing alkyl-chain    substitutions: Relationship of IC.sub.50 to CLOGP               IC.sub.50 (mM)                     prostate               normal    Rx       CLOGP   ca.     glioblastoma                                     melanoma                                            cells    ______________________________________    α-methoxy-PA             2.17    6       5.8     6      ND    PA       2.05    5       4.3     5      12    α-methyl-PA             2.42    2.6     3.8     3.5    12    α-ethyl-PA             2.77    2.1     2.8     2.2     9    PB       2.89    1       1.8     1      ND    4-chloro-PB             3.30    0.75    ND      0.8    ND    4-iodo-PB             3.85    0.36     0.27   0.22   ND    ______________________________________     ND, not determined

                  TABLE 22    ______________________________________    Phenylacetate and analogues containing ring    substitutions: Relationship of IC.sub.50 to CLOGP                IC.sub.50 (mM)                      prostate               normal    Rx        CLOGP   ca.     glioblastoma                                      melanoma                                             cells    ______________________________________    4-Hydroxy-PA              1.78    7.5     10      10     ND    PA        2.05    5       4.3     5      12    4-fluoro-PA              2.17    2.8     4       2.5    ND    2-methyl-PA              2.43    2.5     ND      ND     ND    3-methyl-PA              2.45    2.1     ND      ND     ND    4-methyl-PA              2.47    2.1     ND      ND     ND    4-chloro-PA              2.48    1       0.9     1.2    3    3-chloro-PA              2.54    1.75    1.7     1.5    7    2-chloro-PA              2.56    2.4     2.1     2.5    ND    2,6-dichloro-PA              2.87    1       0.8     1      ND    4-iodo-PA 3.12    0.6     0.9     1.2    ND    1-naphtylacetate              3.16    0.8     0.9     0.8      2.8    ______________________________________     ND, not determined

Further analysis of structure-activity relationships was based on themethod of Hansch and Anderson used for the correlation of the anestheticand metabolic effects of barbiturates with their octanol-water partitioncoefficients Hansch, C., and Anderson, S. M. The structure-activityrelationship in barbiturates and its similarity to that in othernarcotics. J. Med. Chem 10: 745-753, 1967.!. Adaptation of this methodassumes that, if the relationship is simple, it will follow theequation: log 1/C=slope log P+K. Plotting the log 1/IC₅₀ values obtainedwith prostatic cells vs drug CLOGP (FIG. 26) shows that the best fitline is described by the equation: log 1/IC₅₀ =0.89 CLOGP+0.55. Theslope of this line (0.89) is in the range of values found for theanesthetic potencies of a series of barbiturate analogues. Hansch andcolleagues also studied the effect of phenylacetate and its derivativeson plant growth. As shown in FIG. 27, the concentration range and rankorder of inhibition of plant growth by phenylacetate analogues arecomparable to the inhibition of growth of prostatic cancer cells by thissame series of compounds.

While the overall trend of enhanced activity of phenylacetatederivatives with increased lipophilicity is clear, some small deviationsoccur. For both chloro- and methyl-substitutions, the para position ismore potent than the ortho position. In addition, and despite theirnearly equal contributions to lipophilicity, para chloro-substitutionwas more potent than methyl. In contrast to derivatives containing ringor alpha-carbon substitutions, those with blocked carboxyl groupsexhibited a decline in cytostatic activity. The methyl ester ofphenylacetate was about half as active than the free acid (IC₅₀ in DU145prostatic cells 8.8 mM versus 4.1 mM for phenylacetate). The amide formswere also less active than the parent compounds in this experimentalsystem, with IC₅₀ s of 2.0 mM for phenylbutyramide versus 1.2 mM forphenylbutyrate, and 4.8 mM for phenylacetamide versus 4.1 mM forphenylacetate.

Drug Uptake

One possible function of increasing lipophilicity is an increasing easewith which aromatic fatty acids can enter into, and cross the plasmamembrane as well as the membranes of other organelles. The rate ofphenylacetate uptake by tumor cells was compared that of the morehydrophobic analog, naphthylacetate (Table 22). After 10 minutes,relative to phenylacetate more than twice as much naphthylacetate hadentered the glioblastoma U87 cells (uptake of phenylacetic acid was 41%that of naphthylacetic acid) indicating that its movement through theplasma membrane was more than twice as fast as phenylacetate. After 20minutes, the amount of naphthylacetate taken up by the cells was as only26% greater than that of phenylacetate and at 180 minutes theintracellular levels of both compounds were nearly equal, suggestingthat at this time the more rapid influx of naphthylacetic acid wasbalanced by an equally rapid efflux. There was little further uptake andthe concentration of phenylacetate inside and outside the cells wasabout equal indicating that these cells do not actively accumulate mucharomatic fatty acid.

Phenotypic Reversion

In addition to causing selective cytostasis, phenylacetate inducesmalignant cells to undergo reversion to a more benign phenotype. Theeffect of analogs on tumor biology was tested using as a model thehormone- refractory prostatic PC3 cells originally derived from a bonemetastasis. PC3 exhibit several growth characteristics in vitro thatcorrelate with their malignant behavior in vivo, includinganchorage-independent growth (i.e., colony formation in semi-solidagar), and formation of "net"-like structures when plated on areconstituted basement membrane (matrigel). The ability of phenylacetateand representative analogs to bring about loss of such properties issummarized in FIG. 26. Similar to the cytostatic effect, drug ability toinduce reversion to a non-malignant phenotype was highly correlated withthe calculated lipophilicity of the drugs. Of the tested compounds,naphthylacetate, as well as derivatives of phenylbutyrate andphenylacetate with iodo- and chlorine substitutions were found to be themost active on a molar basis. The relative efficacy of the compounds insuppressing anchorage independent growth was confirmed using U87glioblastoma cells (data not shown).

Discussion

The comparative activity of phenylacetate and its analogues against anumber of tumor cell lines suggest that these compounds may form a newclass of therapeutic agents whose effectiveness varies with structure.Improved anticancer activity is achieved if factors controlling theiraction are understood, and toward this end the effects of systematicchanges in structure with changes in activity have been compared. Theoutstanding result is the discovery that: (a) there is a simplerelationship between the lipophilicity of a phenylacetate derivative andits activity against human tumor cells, and (b) the relative potencyobserved with human neoplasms is similar to that documented in plants,indicating that the role of the aromatic fatty acids in growthregulation has been conserved in evolution.

The efficacy of aromatic fatty acids was demonstrated in vitro usingtumor cell lines derived from patients with hormone-refractory prostaticcarcinoma, glioblastomas, and malignant melanoma. Like phenylacetate,several derivatives containing alpha-carbon or ring substitutions allinduced cytostasis and phenotypic reversion at non-toxic concentrations.Changes in tumor biology included reduction in cell proliferation rateand loss of malignant properties such as invasiveness andanchorage-independence. There were, however, significant differences inpotency. When compared to phenylacetate, analogs with naphthyl-,halogen- or alkyl-ring, as well as α-carbon alkyl substitutionsexhibited increased activity, while those with a-methoxy or hydroxylreplacement at the phenyl ring were less effective. Drug potency wascorrelated with the degree of calculated lipophilicity, indicating thatdifferences in efficacy may be due in part to the ease with which theseagents enter into and cross the lipid bilayer of cell membranes. Inagreement, uptake of the more hydrophobic compound, naphthylacetate, wassignificantly faster than that of phenylacetate. At equilibrium (about180 minutes for phenylacetate), however, there were no differences ineither the total intracellular concentration of both compounds, or thelevels inside and outside cells. These results suggest that the rates ofdrug uptake are balanced by proportional rates of efflux, and that theoverall capacity of the cell to retain such compounds is not muchgreater than that of the extracellular milieu.

Although there is a good correlation between drug potency andlipophilicity (see FIG. 26), small deviations within thephenylacetate-related series may give some clues regarding mechanisms ofaction. Halogen substitutions para to the alkylcarboxyl group were foundto increase potency more than those in the ortho position, suggestingthat orientation of the hydrophobic substituent may be important. At thepara position, chlorine had a greater impact on efficacy than a methylgroup despite nearly equal contributions to CLOGP, indicating thatelectronegativity may affect growth inhibitory interactions. Whileα-ethylphenylacetic acid, in which the carboxyl group is crowded by theadjacent ethyl group, was more potent than the parent compound, the morelipophilic analog α-methoxyphenylacetic acid was less active. Theα-methoxyphenylacetic acid is a significantly stronger acid, and thisgreater acidity could be important. Other parameters such as addition ofan aromatic ring to phenylacetate, or an increase in the distancebetween the aromatic nucleus and the carboxyl group did not causeanomalous enhancement or interference in biological activity(naphthylacetate and phenylbutyrate were about as active as would beexpected on the basis of their lipophilicity). The importance of a freecarboxyl group is unclear. The amide forms of phenylacetate andphenylbutyrate, in which the carboxylic group is blocked, were lesscytostatic compared to the parental compounds and failed to induce celldifferentiation (unpublished data). Moreover, phenylacetylglutamine hasno detectable effect on cell growth and maturation. It appears,therefore, that a free carboxyl group may be essential for some aspectsof the antitumor activity of phenylacetate and derivatives.

The correlation between partition coefficients and bioactivity of thearomatic fatty acids is reminiscent of that observed for a large numberof other lipophilic agents. A survey by Hansch and Anderson revealedthat, in a variety of animal tissues, the anesthetic and metaboliceffects of barbiturates corresponded well with their hydrophilicity,having an average slope of about 1 compared to a slope of about 0.67 forlipophilic interaction with protein. It was concluded that the criticalstep in initiating biological activity was entry into the lipid bilayer,probably followed by interaction with membrane proteins. Some of thesubsequently identified targets of barbiturates are indeed, membraneproteins and these include the GABA receptor-chloride in neurons, theATP-K⁺ pump in pancreatic B-cells, and the G-protein that stimulates PLCactivity in leukemic cells. Despite a wide body of literatureimplicating phenylacetate and analogs in growth control throughoutphylogeny, little is known regarding their mode of action. In plants,phenylacetate and naphtylacetate are endogenous growth hormones (auxins)known to stimulate proliferation at micromolar concentrations, whileinhibiting growth at millimolar levels. As growth inhibitors (but notstimulators), the effect of phenylacetate analogues on rapidlydeveloping embryonic plant tissues, like that on human tumor cells, is asimple function of their lipophilicities. These similarities in potency,summarized in FIG. 27, suggest that some of the underlying mechanisms ofnegative growth control may be similar as well.

There is accumulating evidence indicating that phenylacetate andderivatives may act through multiple mechanisms to alter gene expressionand cell biology. At growth inhibitory concentrations, the aromaticfatty acids could alter the pattern of DNA methylation, an epigeneticmechanism controlling the transcription of various eukaryotic genes.Phenylacetate inhibits DNA methylation in plant and mammalian cells, andboth phenylacetate and phenylbutyrate were shown to activate theexpression of otherwise dormant methylation-dependent genes. DNAhypomethylation per se is not sufficient to induce gene expression.Preliminary findings indicate that phenylacetate, phenylbutyrate andseveral analogs activate a nuclear receptor that functions as atranscriptional factor; interestingly, the receptor is a member of asteroid nuclear receptor superfamily, the ligands of which arecarboxylic acids and include well characterized differentiation inducerssuch as retinoids.

In addition to affecting gene transcription, the phenyl-fatty acids mayinterfere with protein post-translational processing by inhibiting themevalonate (MVA) pathway of cholesterol synthesis. MVA is a precursor ofseveral isopentenyl moieties required for progression through the cellcycle, and of prenyl groups that modify a small set of criticalproteins. The latter include plasma membrane G and G-like proteins(e.g., ras) involved in mitogenic signal transduction (molecular weight20-26 kDa), and nuclear envelope lamins that play a key role in mitosis(44-74 kDa). The aromatic fatty acids can conjugate with coenzyme-A,enter the pathway to chain elongation, and interfere with lipidmetabolism in general. Furthermore, compounds such as phenylacetate canassume a conformation resembling mevalonate pyrophosphate and inhibitMVA utilization specifically. It was recently demonstrated thatphenylacetate activity against poorly differentiated mammalian tissues(human glioblastoma cells and the developing fetal brain) is associatedwith inhibition of MVA decarboxylation and a decline in proteinisoprenylation. Rapidly developing mammalian and plant tissues arehighly dependent upon MVA for cell replication. Inhibition of MVAutilization by phenylacetate-related compounds could thus be responsiblein part for their effect documented in such highly divergent organisms.

In conclusion, phenylacetate and analogs appear to represent a new classof pleiotropic growth regulators that might alter tumor cell biology byaffecting gene expression at both the transcriptional and posttranscriptional levels. Phenylacetate and phenylbutyrate have alreadybeen established as safe and effective in treatment of hyperammonemia,and phase I clinical trials in adults with cancer confirmed thatmillimolar levels can be achieved in the plasma and cerebrospinal fluidwith no significant toxicities (discussed herein). However, rather largedoses (300 mg/kg/day or more) are required to achieve potentiallytherapeutic levels. The identified relationship between lipophilicity ofcommercially available analogs and their antitumor activity inexperimental models led us to predict that analogs with greater CLOGPs,e.g., iodo derivatives of phenylacetate and phenylbutyrate, would behighly effective. Indeed, these compounds were found to be the mostpotent aromatic fatty acids yet tested. With this approach, it should bepossible to identify highly effective and safe antitumor agents suitablefor clinical application.

Section L: NAPA and NAPB--Retinoic Acid Combination Treatment

Using the LA-N-5 cell line, NaPA can stimulate the differentiation ofhuman neuroblastoma cells. Furthermore, the results show thatcombination treatment with NaPA and RA results in synergistic anti-tumorand differentiating effects which may be mediated, among othermechanisms, by the ability of NaPA to impact positively on the RAdifferentiation program.

Example 22

LA-N-5 cells--combination of NaPA and RA

Cell culture. The LA-N-5 human neuroblastoma cell line was grown in RPMI1640 medium supplemented with 10% heat-inactivated FCS, HEPES buffer and50 IU/ml penicillin/streptomycin and 1 μg amphotericin (complete medium)as previously described (Sidell, N., Altman, A., Haussler, M. R., andSeeger, R. C. Effects of retinoic acid (RA) on the growth andphenotypic-expression of several human neuroblastoma cell lines. Expl.Cell Res., 148:21-30, 1983). NaPA was obtained from Elan PharmaceuticalResearch Corporation (Gainesville, Ga.). All other chemicals werepurchased from Sigma (St. Louis, Mo.) unless otherwise indicated.All-trans-RA was dissolved in dimethylsulfoxide to a concentration of5×10⁻² mmol and stored at -20 C.

³ H-Thymidine incorporation. Cells (3 to 5×10³ /well) were seeded in24-well plastic tissue-culture dishes (2 cm², Corning, Palo Alto,Calif.) and grown in the absence or presence of the indicatedconcentration of NaPA and/or RA. One μCi ³ H-thymidine/well was added tothe cultures after 6-7 days of growth unless otherwise indicated. Afterincorporation for 12 to 16 hr, cultures were extensively rinsed in PBS,TCA-precipitated for 30 min (10% TCA) and washed in absolute ethanol.Cultures were then solubilized in 1N NaOH, neutralized in 1N HCl and analiquot was counted in Scintifluor (National, Diagnostics, Manville,N.J.) cocktail. Results from typical experiments are expressed as meancpm (±SEM) of triplicate or quadruplicate wells. Each experiment wasperformed at least 3 times with similar results.

Acetylcholinesterase (AChE) activity. Specific AChE activity wasmeasured as a biochemical index of the relative state of differentiationof treated and control LA-N-5 cells (Sidell, N., Lucas, C. A., andKreutzberg, G. W. Regulation of acetylcholinesterase activity byretinoic acid in a human neuroblastoma cell line. Expl. Cell Res.,155:305-309, 1984). For measure of AChE activity, cells were grown in 25cm² tissue culture flasks for 6 days in the absence or presence of theindicated concentrations of NaPA and/or RA, washed twice with isotonicsaline, and harvested by vigorous shaking of the culture flask. Afterremoval of saline, cells were frozen at -20° C., thawed by the additionof ice-cold 10 mM sodium phosphate buffer (pH 7.4) containing 0.5%Triton X-100 (1.5 ml/10⁶ cells), and sonicated for 10 sec. AChE activityin samples of the homogenate was determined photometrically by followingthe hydrolysis of acetylthiocholine as previously described (Sidell, N.,Lucas, C. A., and Kreutzberg, G. W. Regulation of acetylcholinesteraseactivity by retinoic acid in a human neuroblastoma cell line. Expl. CellRes., 155:305-309, 1984). Protein concentrations were determined with aBio-Rad Coomassie protein assay kit using bovine serum albumin as thestandard. Results, in nmoles/hr/mg protein, are expressed asmeans±S.E.M. of triplicate wells in a typical experiment. Allexperiments were repeated at least 3 times.

Northern blot analysis. Total RNA was extracted from harvested cells bythe guanidinium isothiocyanate method and precipitated through a CsClgradient (Chirgwin et al., 1979). The RNA was then stored at -70° untiluse. RNA (20-25 μg) was denatured by glyoxal, fractionated byelectrophoresis through a 1.2% agarose gel at low voltage thentransferred to a Biotrans nylon membrane (ICN, Irvine, Calif.) using apositive pressure transfer apparatus (Stratagene, La Jolla, Calif.). DNAprobes were labelled using a random prime kit and activities of 10⁹cpm/pg were normally obtained (Amersham, Arlington Heights, Ill.).Prehybridization and hybridization medium consisted of 6×standard salinecitrate (SSC), 5×Denhardt's solution, 0.5% SDS and 100 μg/ml salmonsperm DNA. After 16 hrs of incubation, filters were washed once in1×SSC, 0.1% SDS at room temperature for 30 min and 3 times in 0.2% SSC,0.1% SDS at 68°. Autoradiography was performed using Kodak SAR film at-70°. Quantitative densitometry was performed with a scanning laserdensitometer (Biomed Instruments, Irvine, Calif.).

The nuclear retinoic acid receptor-β (RARβ) probe was obtained fromclone RAR-βO as described (Brand, N., Petkovich, M., Krust, A., Chambon,P., de The, H., Marchio, A., Tiollais, P., and Dejean, A. Identificationof a second human retinoic acid receptor. Nature, 332:850-853, 1988).The N-myc specific probe pNB-1 was obtained from American Type CultureCollection. All filters were reprobed with A 600 base EcoRI/BamHIβ-actin fragment in order to confirm comparable loading of samples.

Immunocytology

LA-N-5 cells were plated onto tissue culture chamber slides (Nunc, Inc.,Naperville, Ill.) in complete medium in a humidified 5% CO₂ atmosphere.Cells were allowed to attach overnight then treated with the indicatedconcentrations of NaPA and/or RA. After one week of treatment, cellswere washed in PBS and fixed with 2% paraformaldehyde in PBA at pH 7.0,followed by methanol, at 4° C. for 10 min each. Endogenous peroxidaseactivity was eliminated by incubation in 3% H₂ O₂ in methanol for 10 minat room temperature. Non-specific antigenic sites were blocked by a 30min room temperature incubation with 10% normal goat serum in PBS,followed by a 2 hr incubation with primary antibody at 37° C. Anti-N-mycmonoclonal antibody NCM II 100 (Ikegaki, N., Bukovsky, J., and Kennett,R. H. Identification and characterization of the NMYC gene product inhuman neuroblastoma cells by monoclonal antibodies with definedspecificities. Proc. Natl. Acad Sci. USA, 83:5929-5933, 1986) and anon-binding IgG control monoclonal antibody were used as primaryantibodies in the reactions. Incubation with primary antibody wasfollowed by sequential incubation with biotinylated goat anti-mouseantibody at room temperature for 30 minutes, Z-avidin-peroxidaseconjugates at room temperature for 30 minutes (Zymet Laboratories, Inc.,San Francisco, Calif.), and diaminobenzidine/H₂ O₂ (Sigma) at 0.5 mg/mlin 50 mM Tris pH 8 at room temperature for 10 minutes. Cells were washedfor 15 minutes in PBS between all steps. Nuclear staining intensity wasquantified and analyzed using a Nikon digital image microscope systemand image processing and analysis software from Analytical ImagingConcepts (Irvine, Calif.).

Analysis of protein isoprenylation. Cell cultures were incubated with 10mM phenylacetate and/or 1 μM RA for 24 hrs in complete medium containing5 μM lovastatin. The cells were labeled with RS- 2-¹⁴ C!mevalonate (16μCi/ml, specific activity 15 μCi/mmol) (American Radiolabled Chemicals,Inc., St. Louis, Mo.) during the final 15 hours of treatment. Whole cellproteins were extracted, resolved on 10% SDS-polyacrylamide gels, andstained with Coomassie Brilliant Blue. Gels were then dried and exposedto Kodak X-Omat film for 4 days.

Morphologic differentiation. NaPA induced neurite outgrowth from LA-N-5cells. This effect first became apparent after about 3 days ofcontinuous exposure in culture, with maximal increases in the formationof neurites occurring at NaPA concentrations between 5-10 mM.Concentrations less than 1 mM produced no noticeable morphologiceffects. No decrease in the percentage of viable cells was detected inthe treated cultures as compared with control cultures. However, higherNaPA concentrations (>10 mM) were found to be toxic over extendedculture periods (>7 days). The cell soma were not markedly altered withNaPA treatment, although a slight increase in the number ofintracellular inclusions resembling lipid droplets at NaPAconcentrations greater than 5 mM was observable.

As extensively reported, RA also induced neurite sprouting from LA-N-5cells with maximal effects seen at RA concentrations greater than 1 μM(Abemayor, E., and Sidell, N. Human neuroblastoma cell lines as modelsfor the in vitro study of neoplastic an neuronal cell differentiation.Environ. Health Perspect., 80:3-15, 1989; Sidell, N., Altman, A.,Haussler, M. R., and Seeger, R. C. Effects of retinoic acid (RA) on thegrowth and phenotypic expression of several human neuroblastoma celllines. Expl. Cell Res., 148:21-30, 1983; Lando, M., Abemayor, E.,Verity, M. A., and Sidell, N. Modulation of intracellular cyclic AMPlevels and the differentiation response of human neuroblastoma cells.Cancer Res., 50:722-727, 1990; Sidell, N., Lucas, C. A., and Kreutzberg,G. W. Regulation of acetylcholinesterase activity by retinoic acid in ahuman neuroblastoma cell line. Expl. Cell Res., 155:305-309, 1984). Inthe presence of optimal concentrations of both agents, morphologicdifferentiation of LA-N-5 was profound. Indeed, treatment with 5 mM NaPAplus 1 μM RA induced cellular clustering and neurite bundle recruitmentreminiscent of pseudoganglia formation after only 6-7 days of culture(FIG. 29D). Longer periods with both agents (˜2 weeks) resulted incultures consisting predominantly of large cellular aggregates connectedby thick fascicles of neurites against an elaborate background networkof thin fibers. At this point, very few individual cells (those notassociated with a cell cluster) could be observed.

Proliferation. FIG. 28 shows the dose-dependent effects of NaPAtreatment on ³ H-thymidine incorporation in LA-N-5 cells. It is evidentthat greater than 65% inhibition was achieved at NaPA concentrations of5 mM and above, while little effect was seen below 1.25 mM.Concentrations above 10 mM were not tested since detachment of cellsfrom the flasks suggested significant toxicity.

NaPA and RA were synergistic in their antiproliferative effects onLA-N-5 cells in that combination treatment consistently reduced ³H-thymidine incorporation to lower levels than one would expect from thecombined action of two antiproliferative agents acting throughindependent mechanisms. For example, in FIG. 29, ³ H-thymidineincorporation in the presence of 1.25 mm NaPA was around 90% of controlwhile that with 10⁻⁷ M RA was 70%. Both agents together at theseconcentrations reduced ³ H-thymidine incorporation to 30% of controlwhile 63% would be expected from the two agents acting independently ofeach other (0.90×0.70-63%). Combination treatment at the highestconcentrations tested (NaPA-5 mM; RA-1 μM) resulted in almost completecessation of cell growth while each agent alone produced only a partialeffect after the 6-day assay period.

Acetylcholinesterase (AChE) activity. Concomitant with neuriteoutgrowth, AChE activity increases in LA-N-5 cells induced todifferentiate with a variety of agents including RA (Lando, M.,Abemayor, E., Verity, M. A., and Sidell, N. Modulation of intracellularcyclic AMP levels and the differentiation response of humanneuroblastoma cells. Cancer Res., 50:722-727, 1990; Wuarin, L., Verity,M. A., and Sidell, N. Effects of gamma-interferon and its interactionwith retinoic acid on human neuroblastoma cells. Int. J. Cancer,48:136-144, 1991). To assess whether this biochemical index ofneuroblastoma differentiation was associated with NaPA-induced neuriteoutgrowth, AChE activity was measured in cells treated with variousconcentrations of NaPA for 6-7 days (FIG. 30). As can be seen, AChE wassignificantly increased at 1.25 mM NaPA with maximal increases occurringat 5 mM AChE. In the presence of NaPA and RA, AChE activity was markedlypotentiated over that seen with either agent alone. Thus, as shown inFIG. 30, while AChE activities in either NaPA or RA treated cellsremained below 40 nm/min/mg protein, levels as high as 150 nm/min/mgprotein could be achieved with combination treatments. This activityrepresents the strongest AChE induction yet observed in LA-N-5 cellsusing a variety of differentiating agents, either alone or incombination. Although the absolute values of AChe varied from oneexperiment to another, the pattern was consistent and the results shownin FIG. 30 is representative. Generally, the induced increase in AChEactivity paralleled the extent and complexity of neurite formation inthe LA-N-5 cultures.

Susceptibility of LA-N-5 cells to glutamine depletion. In humans, NaPAcauses depletion of circulating glutamine due to conjugation of theamino acid to form phenylacetylglutamine, an enzymatic reaction known totake place in the liver and kidney (James, M. O., Smith, R. L.,Williams, F. R. S., and Reidenberg, M. The conjugation of phenylaceticacid in man, sub-human primates and some non-primates species. Proc. R.Soc. Lond. B, 182:25-35, 1972). The in vivo reduction in plasmaglutamine levels was mimicked in vitro by culturing cells in thepresence of lowered glutamine concentrations. Glutamine deprivationshowed no significant effect on the growth, morphology, or AChE activityof LA-N-5 cells. Indeed, in the complete absence of exogenous glutamine,LA-N-5 cultures were essentially indistinguishable from those growing inphysiological levels of glutamine. Furthermore, both NaPA- andRA-induced differentiation of LA-N-5 cells were unaffected by theabsence of glutamine. Thus, glutamine depletion by NaPA could notexplain the differentiation-induction effects of this compound on LA-N-5cells.

N-myc expression. Expression of the N-myc oncoprotein has been shown tobe rapidly decreased in LA-N-5 and other neuroblastoma cells duringRA-induced differentiation (Thiele, C. T., Reynolds, C. P., and Israel,M. A. Decreased expression of N-myc precedes retinoic acid-inducedmorphological differentiation of human neuroblastoma. Nature,313:404-406, 1985,RA19). Nuclei of untreated LA-N-5 cells stronglyreacted with the N-myc antibody and showed a marked heterogeneity ofstaining intensity within the culture as previously reported forneuroblastoma cells in general (Ikegaki, N., Bukovsky, J., and Kennett,R. H. Identification and characterization of the NMYC gene product inhuman neuroblastoma cells by monoclonal antibodies with definedspecificities. Proc. Natl. Acad Sci. USA, 83:5929-5933, 1986). Thisheterogeneity was observed by quantitative image analysis of theimmunostained cytoslide preparations. Cells incubated for greater than 3days with NaPA or RA showed marked reductions of N-myc expression asindicated by a decrease in the median relative staining intensity from44 in control cultures to 36 and 29 in NaPA- and RA-treated cultures,respectively (FIG. 31). Combination treatment with both NaPA and RAresulted in a further profound decrease in N-myc levels with a meanrelative staining intensity of 16.

Although NaPA induced a reduction in N-myc protein, no changes wereobserved in N-myc mRNA expression in short term (3-6 day) cultures. Sixday treatment of LA-N-5 cells with 5 mM NaPA (a condition whichconsistently reduced N-myc protein levels) demonstrated no apparentdifference from untreated cultures in N-myc mRNA levels as assessed byNorthern blotting. On the other hand, RA treatment caused a dramaticdecrease in the amount of message produced as previously reported whilecombination treatment resulted in message levels similar to that seenwith RA alone. In contrast to this lack of effect of NaPA on N-myc RNAin short term cultures, longer treatment with NaPA (>8 days) inducedboth a moderate decrease in N-myc mRNA levels and a profound furtherreduction when combined with RA.

Nuclear retinoic acid receptor-β (RARβ) expression. In humanneuroblastoma, as in many other tissues, RARβ shows low constitutiveexpression in untreated cells but is rapidly induced by RA (de The, H.,Marchio, A., Tiollais, P., and Dejean, A. Differential expression andregulation of the retinoic acid receptor α and β genes. EMBO J.,8:429-433, 1989; Wuarin, L., Chang, B., Wada, R., and Sidell, N.Retinoic acid upregulates nuclear retinoic acid receptor-α expression inhuman neuroblastoma. Int. J. Cancer (in press)). RARβmRNA levels weremoderately increased by NaPA, while in the presence of both NaPA and RA,the expression of this receptor was markedly enhanced over that seenwith RA alone. This effect occurred prior to the morphologic or otherphenotypic changes induced by NaPA in LA-N-5 cells.

Effects of NaPA on protein isoprenylation. Active de novo synthesis ofcholesterol and isoprenoids from precursors such as acetyl-CoA andmevalonate (MVA) is an important feature of developing neuronal tissue(Grossi, E., Paoletti, P., and Paoletti, R. An analysis of braincholesterol and fatty acid biosynthesis. Arch. Int. Physiol. Biochem.,66:564-572, 1958). As such, protein prenylation has been shown to be animportant post-translational process critical for normal regulation ofcell growth and differentiation (Marshall, C. J. Protein prenylation: Amediator of protein-protein interactions. Science, 259:1865-1866, 1993;Braun, P. E., De Angelis, D., Shtybel, W. W., and Bernier, L. Isoprenoidmodification permits 2', 3'-cyclic nucleotide 3'-phosphodiesterase tobind to membranes. J. Neurosci. Res. 30:540-544, 1991). As discussedelsewhere herein, NaPA inhibits protein isoprenylation and MVAdecarboxylation in human glioblastoma cells. This effect is alsoobserved in embryonic brain in phenylketonuria, an inborn error ofphenylalanine metabolism which is associated with excessive productionof phenylacetate (Scriver, C. R., and Clow, C. L. Phenylketonuria:epitome of human biochemical genetics. N. Engl. J. Med., 303:1394-1400,1980; Castillo, M., Zafra, M. F. and Garcia-Peregrin, E. Inhibition ofbrain and liver 3-hydroxy-3-methylglutaryl-CoA reductase andmevalonate-5-pyrophosphate decarboxylase in experimentalhyperphenyalaninemia. Neurochem. Res., 13:551-555, 1988). NaPA, but notRA, inhibited protein isoprenylation in LA-N-5 neuroblastoma.Furthermore, in the presence of NaPA and RA, these effects were similarto that induced with NaPA alone.

NaPA induced the differentiation of the LA-N-5 human neuroblastoma cellline as assessed by dose-dependent growth inhibition, neurite outgrowth,increased AChE activity, and reduction of N-myc expression. As discussedherein, NaPA was shown to reduce the malignant phenotype ofpromyelocytic leukemia, prostate cancer, and glioblastoma cells. Theability of NaPA to selectively arrest tumor growth and promotedifferentiation was confirmed using rats with malignant brain tumors(described herein). The data confirms and extends data showingNaPA-induced morphologic differentiation, growth inhibition, andreduction of N-myc protein levels in two other human neuroblastoma celllines (Cinatl, J., Cinatl, J., Mainke, M., Weissflog, A., Rabenau, H.,Kornhuber, B., and Doerr H-W. In vitro differentiation of humanneuroblastoma cells induced by sodium phenylacetate. Cancer Lett.,70:15-24, 1993). NaPA and RA synergized in inducing LA-N-5differentiation. Thus, combination treatment with NaPA and RA atconcentrations that were saturating in terms of a differentiationresponse with each agent alone caused further marked enhancement of allparameters measured. Furthermore, at all concentrations, combinationtreatment consistently induced LA-N-5 differentiation to a level thatwas greater than would be expected from two agents acting independentlyof each other. Indeed, the combined effects of both agents represent thestrongest differentiation response yet observed by neuroblastoma cells,both in terms of number of cells responding and maturational levelachieved.

These results suggest that NaPA and RA do not work through coincidentalpathways of action but that their pathways must, nevertheless, intersectat some level to synergistically potentiate cellular differentiation andgrowth arrest. This contention was supported by the finding that NaPAmarkedly enhanced RA induction of RARβ mRNA levels in LA-N-5 cells.Upregulation of this receptor has been shown to be a necessary event inRA-induced differentiation of embryonal carcinoma cells (Kruyt, F. A.,van der Brink, C. E., Defize, L. H., Donath, M. J., Kastner, P.,Kruijer, W., Chambon, P., and van der Saag, P. T. Transcriptionalregulation of retinoic receptor beta in retinoic acid-sensitive andresistant P19 embryocarcinoma cells. Mech. Dev., 33:171-178, 1991), andaberrant expression or regulation of RARβ has been suggested in thepathogenesis of certain lung and hepatocellular carcinoma (Nervi, C.,Volberg, T. M., George, M. D., Zelent, A., Chambon, P., and Jetten, A.M. Expression of nuclear retinoic acid receptors in normaltrachiobronchial cells and in lung carcinoma cells. Expl. Cell Res.,195:163-170, 1991; Hu, L., Crowe, D., Rheinwald J., Chambon P., andGudas L. Abnormal expression of retinoic acid receptors and keratin 19by human oral and epidermal squamous cell carcinoma cell lines. CancerRes., 51:3972-3981, 1991), and head and neck tumors (de The, H.,Marchio, A., Tiollais, P., and Dejean, A. A novel steroid thyroidhormone receptor-related gene inappropriately expressed in humanhepatocellular carcinoma. Nature, 330:667-670, 1987). Thus, bymodulating expression of RARβ, NaPA might impact on the RAdifferentiation program in such a way as to lead to enhanced retinoidactivity. However, NaPA can induce LA-N-5 differentiation in culturesconsisting of delipidated FCS and hence devoid of serum retinoids.Therefore, an ability to alter cellular responses to available retinoidconcentrations cannot adequately account for both the direct action ofNaPA, and its synergy with RA. In this regard, it is possible that thedirect action of NaPA in inducing differentiation is mediated bydifferent intracellular events than those responsible for itsinteraction with RA.

A consistent biochemical change induced by NaPA, but not by RA, is adecline in protein isoprenylation. This effect occurs within 24 hrs ofLA-N-5 treatment with NaPA, preceding changes in DNA synthesis anddifferentiation. As recently documented in malignant glioblastomas,inhibition of protein prenylation by NaPA is due primarily to a decreasein MVA decarboxylation, a key step regulated by MVA-5-pyrophosphatedecarboxylase. MVA is a precursor of several isopentenyl moietiesrequired for progression through the cell cycle such as sterols,dolichol, the side chains of ubiquinone and isopentenyladenine, andprenyl groups that modify a small set of proteins (Marshall, C. J.Protein prenylation: A mediator of protein-protein interactions.Science, 259:1865-1866, 1993; Braun, P. E., De Angelis, D., Shtybel, W.W., and Bernier, L. Isoprenoid modification permits 2',3'-cyclicnucleotide 3'-phosphodiesterase to bind to membranes. J. Neurosci. Res.30:540-544, 1991; Goldstein, J. L., and Brown, M. S. Regulation of themevalonate pathway. Nature, 343:425-430, 1990). The latter includeplasma membrane G and G-like proteins (e.g. ras) involved in mitogenicsignal transduction (molecular weight 20-26 kDa), and nuclear envelopelamins that play a key role in mitosis (44-74 kDa). The present studiesshow a decline in prenylation of corresponding proteins in NaPA-treatedLA-N-5 cells, although the specific targets have yet to be identified.Lovastatin, a competitive inhibitor of 3-hydroxy-3-methylglutarylcoenzyme a reductase (which catalyzes the synthesis of MVA fromacetyl-CoA) has also been shown to induce the differentiation ofneuroblastoma cells as described elsewhere herein (Maltese, W. A., andSheridan, K. M. Differentiation of neuroblastoma cells induced by aninhibitor of mevalonate synthesis: Relation of neurite outgrowth andacetylcholinesterase activity to changes in cell proliferation andblocked isoprenoid synthesis. J. Cell. Physiol., 125:540-558, 1985). Theexperience with NaPA and lovastatin suggested that growth arrest andmaturation may not be directly related to a decline in cholesterol,dolichol, or ubiquinone, but rather due to depletion of isoprenoidcompounds essential for protein processing and maintenance of themalignant phenotype (Maltese, W. A., and Sheridan, K. M. Differentiationof neuroblastoma cells induced by an inhibitor of mevalonate synthesis:Relation of neurite outgrowth and acetylcholinesterase activity tochanges in cell proliferation and blocked isoprenoid synthesis. J. Cell.Physiol., 125:540-558, 1985). Since isoprenylation was not affected byRA treatment, it is unlikely that inhibition of this process by NaPA isa direct result of any NaPA-induced changes in the RA differentiationprogram (e.g. an increase in RARβ expression). Thus, reduction ofprotein isoprenylation by NaPA reflects either early events leading to(rather than a consequence of) alterations in the retinoid pathway ofaction or a totally independent effect that could mediate some of thecellular responses to NaPA apart form its interaction with RA.

The effect of NaPA on N-myc mRNA levels in LA-N-5 cells was especiallyintriguing. In short term (3-6 day) cultures, NaPA had no apparenteffects on N-myc mRNA levels, either alone or in the presence of RA eventhrough N-myc protein was found to be decreased. However, a significantdecline in N-myc RNA expression was seen in NaPA-treated cultures afterlonger-term treatment. This finding suggests a posttranscriptional levelof regulation by NaPA that ultimately results in highly differentiatedcells expressing lowered levels of N-myc RNA. Indeed, in the combinedpresence of NaPA and RA for 10 days, the decrease in N-myc is dramatic,with very little mRNA detectable by Northern blotting. In studies inHL60 cells (see supra), NaPA caused a rapid decline in amounts of c-mycmRNA, which occurred within 4 hr of treatment, preceding phenotypicchanges associated with differentiation. Thus, alteration by NaPA ofc-myc in HL60 cells and N-myc in LA-N-5 cells may involve differentmechanisms of action. Similarly, modulation of these two oncogenes by RAhas been found to have distinct mechanisms in that down-regulation ofN-myc was shown to be at the level of transcription (Wada, R. K.,Seeger, S. C., Reynolds, C. P., Alloggiamento, T., Yamashiro, J. M.,Ruland, C., Black, A. C., and Rosenblatt, J. D. Cell type-specificexpression and negative regulation by retinoic acid of the human N-myconcogene with rapid progression of neuroblastoma. N. Engl. J. Med.,313:111-116, 1985) while that of c-myc is posttranscriptional (Doty, C.,Kessel, M., and Gruss, P. Post-transcriptional control of myc and p53expression during differentiation of the embryonal carcinoma cell lineF9. Nature, 317:636-639, 1985). In any case, since expression of N-mychas been positively correlated with tumor progression and poor prognosis(Seeger, R. C., Brodeur, G. M., Sather, H., Dalton, A., Siegel, S. E.,Wong, K. Y., and Hammond, D. Association of multiple copies of the N-myconcogene with rapid progression of neuroblastoma. N. Engl. J. Med.,313:111-116, 1985; Grady-Leopardi, E. F., Schwab, M., Ablin, A. R., andRosenau, W. Detection of N-myc oncogene expression in humanneuroblastoma by in situ hybridization and blot analysis: relationshipto clinical outcome. Cancer Res., 46:3196-3199, 1986), the dramaticreduction in the levels of this oncoprotein seen in the presence of NaPAand RA underscores the potentially increased clinically efficacy of thiscombination treatment.

An interaction between NaPA and RA in inducing cellular differentiationand growth inhibition does not appear to be a unique phenomenon forLA-N-5 cells. NaPA-induced maturation of human leukemia, breastcarcinoma, and malignant melanoma can be enhanced by the addition of lowdoses of RA (Samid, D., Shack, S., and Sherman, L. T. Phenylacetate: Anovel nontoxic inducer of tumor cell differentiation. Cancer Res.,52:1988-1992, 1992). Furthermore, Gorski et al. noted synergistic growthinhibition with NaPA and RA of human rhabdomyosarcoma cells (Gorski, G.K., Donaldson, M. H., and McMorrow, L. E. Synergistic inhibition ofhuman rhabdomyosarcoma cells by sodium phenylacetate and tretinoin. InVitro Cell Dev. Biol., 29A:189-191, 1993). Experiments indicated thatNaPA induced growth inhibition and neurite outgrowth from the LA-N-2 andIMR32 human neuroblastoma cell lines, and that these effects werepotentiated with RA. Taken together, these findings suggest that thesynergistic phenomena detailed here with LA-N-5 cells may be a generalfeature of NaPA/RA treatment or many tumor cell types.

Example 23

Antiproliferative effect on Neuroblastoma Cell Lines

Cell lines. In a related experiment, seven human neuroblastoma celllines were used with varying characteristics relating toneurotransmitter phenotype, N-myc amplification, N-ras and p53expression, neurocrest lineage response, and sensitivity to retinoicacid-induced differentiation. Table 23 below summarizes some importantcharacteristics of the seven lines.

                  TABLE 23    ______________________________________    Neuroblastoma Cell Lines                       Changes    Number of    Cell   Neurotransmitter                       induced by N-myc  Expression    Line   phenotype   retinoic acid                                  copies of p53    ______________________________________    SK-N-AS           cholinergic resistant  1      .sup.  n.d..sup.a    LA-N-5 mixed       neuronal   50     1+.sup.c                       differentiation                       (4+).sup.b    LA-N-2 cholinergic neuronal   25     n.d.                       differentiation                       (2+)    SK-N-SH-           adrenergic  schwannian 1      + and    F                  transformation    ras.sup.d    SK-N-SH-           adrenergic  neuronal   1      + and    N                  differentiation   ras                       (3+)    LA-N-6 adrenergic  growth     1      n.d.                       inhibition only    Lan-1- adrenergic  growth     100    15N                inhibition only    ______________________________________     .sup.a not determined     .sup.b relative differentiation response     .sup.c relative expression of p53     .sup.d also possesses a mutationally activated Nras

The prototype and best characterized line is LA-N-5, which, like themajority of human neuroblastoma cell lines, contains amplified copies ofN-myc and is sensitive to differentiation induction by retinoic acid. Assuch, LA-N-5 was the best defined model system to address specificquestions relating to the reversibility of PA and PB and therelationship of treatment duration to agent concentration.

Antiproliferative effects of PA and PB on human neuroblastoma cells.FIG. 32 shows the concentration-dependent effects of PA and PB onincorporation of ³ H!thymidine in the seven cell lines after 7 days oftreatment. As can be seen, in six of seven lines, greater than 95%inhibition was achieved at the highest PB concentration tested (4 mM)with the same cell lines exhibiting greater than 70% inhibition at 2 mMPB. One line, LA-N-6, was found to be less sensitive to PB by showinglittle growth inhibition at concentrations less than 4 mM.

In all cases, the neuroblastoma lines were less sensitive to growthinhibition by PA than by PB. Table 24 extrapolates from FIG. 32, theconcentrations of PA and PB that can induce a 50% growth inhibition inthe cell lines (GI₅₀). As is seen, the three lines least sensitive to PA(LA-N-2, SK-N-SH-N, and LA-N-6) were also the least sensitive to PB.However, the converse was not true; SK-N-AS was the most sensitive togrowth inhibition by PA but was not the most sensitive to PB. No obviouscorrelation was seen between the general characteristics of the celllines as shown in Table 24 and their growth inhibition by PA or PB.

                  TABLE 24    ______________________________________    Growth Inhibition of Cell Lines    Cell Line     GI.sub.50 (mM) PA                             Values of PB    ______________________________________    SK-N-AS       1.8        0.8    LA-N-5        3.5        0.6    LA-N-2        10.0       1.6    SK-N-SH-F     4.5        <0.5    SK-N-SH-N     8.5        1.5    LA-N-6        10.0       3.0    Lan-1-15N     4.5        0.5    ______________________________________

These results indicate that, as a histological group, humanneuroblastoma cells are very-sensitive to growth inhibition by PB andPA. In observing morphologic changes that occurred during the PA and PBtreatments, general features of neurite extension, and cellularclustering in all of the cell lines were also noted. Thus, although PB-and PA-induced neurotransmitter changes in the various lines have notyet been quantitated, the morphologic changes observed suggest thatneuronal differentiation is a general feature in the response of humanneuroblastoma cells to PB and PA.

Dose- and time-dependent effects of PB and PA on neuroblastoma cells.LA-N-5 cells were treated with PB and PA at doses ranging from 0.5 to 4mM and 1.25 to 10 mM respectively, and ³ H!thymidine incorporation (as ameasure of growth) and acetylcholinesterase (as a measure ofdifferentiation) were assessed over the next 8 days. As seen in FIG.33A, cells incubated with 2 or 4 mM PB demonstrated a decrease in ³H!thymidine incorporation as early as two days after the start ofculturing while those incubated with 0.5 and 1 mM PB showed significantinhibition only after four days of treatment. The highest concentrationsof PB (4 mM) seemed to induce a cytotoxic effect which was reflected bya progressive decrease in cell viability (as assessed by trypan blueexclusion) starting on the fourth day of culturing. No decrease in thepercentage of viable cells was detected in cultures treated with theother concentrations of PB. A similar inverse relationship between agentconcentration and treatment time needed to show significant growthinhibition was noted for PA (FIG. 33B). However, in agreement with FIG.32, PA was less effective than PB in that significant growth inhibitionwas first noted only after 3 days at the highest (10 mM) PAconcentration tested. It took at least four days for all otherconcentrations of PA to induce growth inhibition of LA-N-5 cells.

Induction of AChE activity by PB and PA generally occurred quicker thandid growth inhibition induced by these agents (FIG. 34). LA-N-5 cellscultured with all concentrations of PB showed a dose-dependentenhancement in AChE activity starting on the very first day of treatmentand progressively increased to day 4. After this time (day 6 and 8),AChE levels generally leveled off with the exception of a sharp decreaseseen in the presence of 4 mM PB. This decrease probably reflected thereduced viability of these cultures as described above. With PA, AChEactivity in LA-N-5 cultures also showed a time- and dose-dependentincrease, but this effect progressed with all concentrations up to thelast day of measurement (day 8). Studies on the reversibility of PA andPB treatment (below) suggest that AChE levels continue to increase inPA-treatment cultures up to 2 weeks or beyond.

Reversibility of PB and PA effects on growth and AChE activity. LA-N-5cells were cultured with either solvent control, 2 mM PB, or 5 mM PA for6 days, then washed and refed with either control medium or mediumcontaining the agent at the original treatment concentration. ³H!thymidine incorporation (FIG. 35) and specific AChE activity (FIG. 36)were then assessed starting 1 day after washing (posttreatment) andvarious days thereafter. As can be seen in FIG. 35, up to 7 days aftertreating cells with PA, ³ H!thymidine incorporation in cultures refedwith control medium continued to slowly increase at basically the samerate as those refed with PA. However, after 1 week posttreatment, cellsrefed in control medium showed a higher rate of proliferation than thosecultured in the continuous presence of PA. A similar slow reversibilitywas seen with PB in that very little growth was seen for up to 7 daysposttreatment in cultures refed with either control or PB-containingmedium, but following this time period the two growth curves diverged.The slow steady increase in ³ H!thymidine incorporation seen in thecontinuous presence of 5 mM PA for posttreatment periods even up to 14days (20 days total treatment time) suggests either that thisconcentration of PA is not totally suppressing cell growth or thatdiscrete populations of LA-N-5 cells with variable resistance to theagent are present in the culture. Although it is difficult todistinguish between these two possibilities, the initial impression byobserving the gross dynamics of cell populations during culture is thatsituations are true. In contrast, cells grown in the continuous presenceof PB for up to 20 days did not show any growth, suggesting a morecomplete suppression than with PA and/or a lack of resistantpopulations. However, even the profound antiproliferative effect of PBappeared to be somewhat reversible under the conditions of thisexperiment.

Measurements of AChE activity following the 6 days of culturing with theagents supports a conclusion that the effects of both PA and PB on thegrowth and differentiation of LA-N-5 is reversible under the conditionsimposed. As seen in FIG. 36, the increased AChE activity originallyinduced by PA and PB returned to baseline values after 1 weekposttreatment. In those cultures refed with PB, AChE activity remainedelevated to a more or less constant level. On the other hand, incultures refed with PA, specific AChE activity continued to increaseeven up to the 7-day posttreatment time point, eventually reachinglevels greater than that achieved with PB. This latter observationsuggests that induction of AChE (and presumably differentiation) by PAis a relatively slow process and may require many weeks of continuousexposure to the drug before its full differentiation-inducing potentialis realized. Whether or not such longer treatment times might result inirreversible differentiation is presently unknown.

The results of the experiments performed to date have established thefollowing: 1) Human neuroblastoma cells are, as a histologic group,sensitive to growth inhibition and morphologic differentiation by PA andPB. These effects show a 3- to 10-fold greater sensitivity to PB than toPA and are not correlated with any known characteristics of the cellssuch as neurotransmitter phenotype, N-myc amplification and expression,or susceptibility to differentiation by retinoic acid. 2) Using LA-N-5neuroblastoma cells as a model system, differentiation by PB and PA wasfirst evident after one day of treatment at the higher concentrationstested, reached plateau levels of induction after 4 days in the case ofPB but continued to progress for at least 2 weeks in the case of PA. 3)Differentiation by PA and PB following a 6-day treatment protocol wasreversible. The possibility that longer treatments may induceirreversible differentiation is not suggested in the case of PB sinceits effects on the cells seemed to plateau after around 4 days ofexposure. However, since induction by PA progresses for at least 2 weeksto eventually reach a greater stage of maturation than with PB (asassessed by specific AChE activity), future PA reversibility experimentswith longer exposure times would appear to be warranted.

Based on the above-described discovery of the synergistic activitybetween retinoic acid (RA) and PA in inducing neuroblastomadifferentiation, the idea that combination treatment with thesecompounds holds the most potential for their positive therapeutic use iscentral. Indeed, the interaction of PA with retinoids might be onereason by PA appears to be a much more potent anticancer agent in invivo versus in vitro models. Thus, in vitro cultures have a limitedsupply of endogenous retinoids that are carried along with the fetalcalf serum and are degraded during shelf storage, heat inactivation,light exposure, and culturing. On the other hand, serum retinoidconcentrations are tightly regulated in vivo and remain relativelyconstant (in the serum, retinol levels are in the 1 μM range with RAlevels being around 0.01 μM). As such, the addition of exogenousretinoids to in vitro culture systems and the study of the interactionof these compounds with PA and its derivatives may actually be a muchmore "physiologic" model for understanding potential single agentapplications in vivo.

Example 24

NaPA in combination with Flavonoids and Lignins

Futhermore, PA and its analogs may similarly also be used in combinationwith flavonoids and lignins, as well as with retinoids as describedabove. For instance, FIG. 37 shows that PA and apigenin (a flavonoid)act in a synergistic manner to suppress the growth of human prostaticcarcinoma PC3 cells. Similar results were observed when PA was used incombination with 9-cis-retinoic acid. The 9-cis-RA (and its precursors,including all-trans-RA as discussed above) activate the nuclear receptorRXR, which is required for the stimulation of PPAR (described herein andin the copending application) by PA and its analogs.

PC3 cells were treated with 1) 10 μM EtOH (control), 2) PA 4mM, 3)apigenin 10 μM, 4) apigenin 10 μM in combination with PA 4 mM, 5)9-cis-RA 2 μM and 6) 9-cis-RA 2 μM in combination with PA 4 mM. Thecombination therapies showed significant, potentiated reduction(apigenin/PA approximately 50% and 9-cis-RA/PA approximately 50%) in PC3cell survival rates. In addition, the flavonoid quercetin, which isknown to block the efflux of PA from plant cells, may be used incombination with PA to enhance the therapies described herein.

Section M: NaPA and NaPB--Lovastatin Combination Treatment

Malignant gliomas are highly dependent on the mevalonate (MVA) pathwayfor the synthesis of cholesterol and intermediates critical to cellreplication. Targeting MVA synthesis and/or utilization thus inhibitstumor growth without damaging normal brain tissues, in which the MVApathway is minimally active. Human gliobastoma cells were found to beuniquely vulnerable to lovastatin (LOV) and sodium phenylacetate (NaPA)which act as inhibitors of the key regulatory enzymes HMG-coA reductaseand MVA-PP decarboxylase, respectively.

Example 25

NaPA combination therapies with vastatins such as lovastatin

In vitro testing. Monotherapies of both LOV (see Table 25) and NaPA areeffective (induction of cytostasis and phenotypic reversion) againstgliomas in laboratory models and in man (described herein). However,when combined, the two drugs act synergistically to suppress glioma cellproliferation and induce reversion to a benign phenotype. Specifically,treatment of human glioblastoma A172 cells with pharmacologicallyachievable, yet suboptimal concentrations of LOV (0.1-0.5 μM) combinedwith NaPA (1-3 mM) resulted in: (a) complete arrest of tumor cellreplication (see FIG. 61); (b) over 90% decline in invasive capacity(see FIG. 34); and (c) profound inhibition of expression of TGFβ2,coding for a 12.5-kD protein implicated in glioma autocrine growth,angiogenesis, and tumor-induced immunosuppression. Synergy between NaPAand LOV could be due to the ability of each to block the MVA pathway atdistinct regulatory sites, leading to inhibition of proteinisoprenylation. Furthermore, NaPA may further induce tumor cytostasisand differentiation through additional mechanisms such as DNAhypomethylation, activation of nuclear receptors involved in growthcontrol and glutamine depletion.

                  TABLE 25    ______________________________________    Gliomas are Uniquely Vulnerable to Lovastatin Treatment                   Conc. of LOV required for 50% or more    Cell Type      inhibition of tumor replication    ______________________________________    Glioblastoma (A172, U87,                   0.2-2        μM    U251)    Melanoma (1011)                   20           μM    Lung Adenocarcinoma (A549)                   7            μM    Prostate Cancer (PC3)                   5-10         μM    Neuroblastoma (C1300)                   25           μM.sup.a    EJ Bladder Carcinoma                   16           μM.sup.b    ______________________________________     .sup.a Maltese et al., 1985.     .sup.b Sebti et al., 1991.

In vivo testing. Lovastatin was administered orally to 13 patients withrefractory grade 3 and 4 astrocytomas at doses ranging from 30 to 35mg/kg/day for seven consecutive days every four weeks. Activity wasdocumented in four patients: 1 partial and 1 minor response, as well asdisease stabilization for over six months in 2 additional patients.Performance status improved from 20 to 40% on Karnofsky's scale. NaPA(dose range 15 to 40 grams/24 h) was administered by continuous orinterrupted intravenous infusions (CIVI) to 12 patients with similarhistologies and clinical courses. Four patients exhibited clinicalimprovement: 2 minor responses and 2 disease stabilizations withsignificant improvement in performance status (1 for over 8 months, theother for over 1 month). Both modes of administration were associatedwith clinical activity (daily serum concentrations, mean±S.D.: 174±97μg/ml).

Early clinical experience with NaPA and LOV, individually, showedactivity in patients with high grade gliomas at well tolerated doses.Interestingly, one patient who failed to respond to LOV, showedobjective and clinical improvement upon treatment with NaPA, indicatingthat there may be no cross-resistance to these drugs. NaPA and LOVapparently do not have overlapping toxicities. While the dose-limitingtoxicity of NaPA (serum concentration over 900 μg/ml) is reversible CNStoxicity, rhabdomyolysis-induced myopathy was seen with LOV (at 35mg/kg/day) (readily controlled by oral ubiquinone supplementation). Thusadministration of NaPA in combination with LOV is beneficial to gliomapatients without signficant side effects or risk of toxicity.

Section N: NaPA and NaPB Effects on Melanoma Cell Lines

The increased incidence of melanoma and the poor responsiveness ofdisseminated disease to conventional treatments call for the developmentof new therapeutic approaches. Phenylacetate, a nontoxic differentiationinducers can suppress the growth of other neuroectodermal tumors, i.e.,gliomas, in laboratory models and in man. This led to exploration of theefficacy of phenylacetate and related aromatic fatty acids in malignantmelanoma. Phenylacetate and phenylbutyrate were found to: (a) induceselective cytostasis and maturation of cultured human melanoma cells,(b) modulate the expression of genes implicated in tumor metastasis(collagenase type I, TIMP) and immunogeneicity (HLA class I);.and, (c)enhance the efficacy of other agents of clinical interest, includingretinoids, interferon alpha, suramin, and 5-aza-2'-deoxycytidine.Reflecting on the phenotypic heterogeneity of melanoma, the degree ofbiological alteration induced by phenylacetate/phenylbutyrate variedsignificantly among the tumor cell lines tested. While losing invasivecapacity and tumorigenicity in athymic mice, poorly differentiated cellsexhibited only a marginal changes in morphology, remained amelanotic,and resumed growth after treatment was discontinued. By contrast,treatment of melanoma cells that were in a more advanced stage ofmaturation resulted in profound alterations in cell growth, morphologyand pigmentation consistent with terminal differentiation.Concentrations of phenylacetate and phenylbutyrate affecting tumor cellbiology in vitro have been achieved in humans with no significanttoxicities, suggesting clinical efficacy of these drugs in the treatmentof malignant melanomas.

Sodium phenylacetate has been described herein as a nontoxicdifferentiation inducer (Samid, D., Shack, S., Sherman, L. J. (1992):Phenylacetate: a novel nontoxic inducer of tumor cell differentiation.Cancer Research 52:1988-1992). Phenylacetate, a common metabolite ofphenylalanine, regulates cell growth in diverse organisms throughoutphylogeny (Kishore, G., Sugumaran, M., Vaidyanathan, C. S. (1976):Metabolism of DL-phenylalanine by Aspergillus niger. J. Bacteriol.128:182-191). At millimolar concentrations, phenylacetate selectivelysuppresses the growth of poorly differentiated plant, rodent, and humantissues (Wightman, F., Lighty, D. L. (1982): Identification ofphenylacetic as a natural auxin in the stoots of higher plants. Physiol.Plant. 55:17-24). Cancerous cells, which are highly reminiscent ofrapidly developing, immature tissues, are likewise vulnerable. NaPApromotes differentation of human leukemic cells (Samid, D., Yeh, A.,Prasanna, P. (1992): Induction of erythroid differentiation and fetalhemoglobin production in human leukemic cells treated withphenylacetate. Blood 80:1576-1581) and brings about reversal ofmalignancy of various solid tumor cell lines, includinghormone-refractory prostatic carcinoma, glioblastoma, neuroblastoma(Jindrich Cinatk, Jaroslav Cinatl, Marion Mainke, Albrecht Weibflog,Holger Rabenau, Bernhard Kornhuber, Hans-Wilhelm Doerr (1993): In vitrodifferentiation of human neuroblastoma cells induced by sodiumphenylacetate. Cancer Letters 70:15-24), and rhabdomyosarcoma (GrzegorzK. Gorski, Lydia E. McMorrow, Milton H. Donaldson (1993): Letter to theeditor: synergistic inhibition of human rhabdomyosarcoma cells by sodiumphenylacetate and tretinoin. In Vitro Cell. Dev. Biol. 29:189-191). Thecentral observation is that NaPA is active in tumor models atconcentrations that can be achieved in children and adults with nosignificant adverse effects (Brusilow, S. W., Danney, M., Waber, L. J.,Batshow, M., Burton, B., Levitsky, L., Roth, K., McKeethren, C., Ward,J. (1984): Treatment of episodic hyperammonemia in children with inbornerrors of urea synthesis.

N. Eng. J. Med. 310:1630-1634; Simell, O., Sipila, I., Rajantie, J.,Valle, D. L., Brusilow, S. W. (1986): Waste nitrogen excretion via aminoacid acylation: benzoate and phenylacetate in lysinuric proteinintolerance. Pediatric Res. 20:1117-1121). Moreover, the activityagainst central nervous system (CNS) tumors, observed in cell culturesand rat models, has recently been confirmed in patients with advanceddisease. The responsiveness of brain tumors that are refractory toconventional systematic therapies suggested that other neoplasms ofneuroectodermal origin, including melanoma, may be susceptible as well.

Phenylacetate induces selective tumor cytostatis and phenotypicreversion of human melanoma cells when used at pharmacological nontoxicconcentrations. Moreover, the present studies demonstrate for the firsttime, that phenylbutyrate, a precursor of phenylacetate, is a potentinducer of tumor cytostatis and differentiation.

Example 26

NaPA treatment of melanoma cells

Cell Cultures and Reagents. The SKMEL 28, G361 and RPMI melanoma celllines were from the American Type Culture Collection (ATCC, Rockville,Md.). A375 was a gift from J. Fidler (M. D. Anderson, Houston, Tex.).Melanoma lines 624 mel, 501 mel, 888 mel and 1011 mel were establishedfrom patients seen at the Surgery Branch NCI. These tumor cell lines aswell as normal human melanocytes were kindly provided by J. Weber (NCI,Bethesda Md.). All tumor cultures were maintained in RPMI 1640supplemented with 10% heat-inactivated fetal calf serum (GibcoLaboratories), antibiotics, and 2 mM L-glutamine. Primary melanocytecultures were maintained in Melanocyte Basal Medium (Clonetics, SanDiego, Calif.). The sodium salts of phenylacetic acid (NaPA) andphenylbutyric acid (NaPB) were provided by Elan Pharmaceutical ResearchCorp., Gainesville, Ga.

PAG was synthesized by the reaction of phenylacetychloride withglutamine (Thierfelder, H., and Sherwin, C. P. (1914):Phenylacetyl-Glutamin, ein Stoffwechsel-Product des MenschliechenKorpers (o") nach Eingabe von Phenyl-essigsaure (a"). Ber. chem. Ges.47:2630-2634). Briefly, 7.5 g glutamine (Aldrich, Milwaukee, Wis.) and8.4 g NaHCO₃ were added to 200 ml H₂ O, and the mixture was adjusted topH 10. While stirring vigorously, 7.5 g of phenylacetylchloride(Milwaukee, Wis.) was added dropwise over the course of 1 hr. When thelast of the phenylacetylchloride had dispersed, the solution wasadjusted to pH 2, extracted twice with hexane, and taken to dryness. Thedried powder was rinsed with hexane and dissolved in a minimum ofboiling H₂ O. Crystallized PAG having the convert melting range and alean HPLC profile was confirmed. Suramin was from Mobay Chemical Corp.(New York, N.Y.). Interferon-alpha (Referon-A) was purchased fromHoffmann La-Roche Inc. (Nutley, N.J.). 5AzadC and all-trans-RA were fromSigma (St. Louis, Mo.).

Analysis of Cell Proliferation and Viability. Growth rates weredetermined by cell enumeration using a hemocytometer followingdetachment with trypsin/EDTA, and by an enzymatic assay using 3-4,5-dimethylthiazol-2-yl!-2,5-diphenyltertrazolium bromide (MTT) (Alley,M. C., Scudiero, D. A., Monks, A., Hursey, M. L., Czerwinski, M. J.,Fine, D. L., Abbott, B. J., Schoemaker, R. H., Boyd, M. R. (1988):Feasibility of drug screening with panels of human tumor cell linesusing a microculture tetrazolium assay. Cancer Res. 48:589-601). The twoassays produced essentially the same results. DNA synthesis wasdetermined by metabolic labeling with ³ H! deoxythymidine (6.7 Ci/mmol)(New England Nuclear). Cell viability was assessed by trypan blueexclusion.

Immunocytochemistry. Cells were immunostained with anti-vimentinmonoclonal antibodies using Dako PAP kit K537 (Dako Corporation, CA).

Invasion Through Matrigel. The ability of tumor cells to degrade andcross tissue barriers was assessed by an in vitro invasion assay thatutilizes matrigel, a reconstituted basement membrane. Quantitativeanalysis was performed using Biocoat Matrigel invasion chamber (BectonDickinson Labware, Bedford, Mass.) according to manufacturer'sinstructions.

Tumor Formation in Athymic Mice. Cells (5×10⁵ cells per site) wereinjected s.c. into 4-6 week old female athymic nude mice (Division ofCancer Treatment, NCI animal Program, Frederick Cancer ResearchFacility). The number and size tumors were recorded after 4 weeks.

Quantitation of Melanin Production. Melanin content was measured by thecalorimetric method described by Whittaker (Whittaker, J. R. (1963):Changes in melanogenesis during the dedifferentiation of chick retinalpigment cells in cell culture. Dev. Biol. 8:99-127). Briefly, tumorcells were plated at 1-2×10⁶ cells per point) by the addition of 0.5 ml.of deionized water with 2 cycles of freezing and thawing. Perichloricacid was added to a final concentration of 0.5N and the suspension waskept on the ice for 10 min, then centrifuged at 15,000 rpm for 5 min.The pellets were extracted twice more with 0.5N HCl₄ followed by twoextractions with a cold mixture of ethyl alcohol:ethyl ether (3:1, v/v)and final extraction with ethyl ether. The pellets were air dried, 1 ml0.85N KOH was added, and then dissolved by heating to 100° C. for 10min. After insoluble residue was pelleted and the supernatant was cooledto room temperature and the absorbance at 400 nm was read in a doublebeam spectrophtometer (UV-160A, Shimadzu Corporation, Kyoto, Japan). Astandard curve was constructed by using synthetic melanin (Sigma)dissolved in hot KOH at concentrations ranging from 5 to 150 μg/ml. Therelative melanin content is expressed as the absorbance at 400 nm per5×10⁶ cells/ml.

Northern Blot Analysis. Messenger RNA was extracted from treated andcontrol cells by Invitrogen mRNA isolation kit (San Diego, Calif.).Samples (5 μg/lane) were electrophoresed through 1% agarose/formaldehydegels and blotted onto nytran membranes (Schleicher & Schuell, Keene,N.H.), UV-cross-linked, and hybridization with ³² P-labeled specificprobes. Tyrosinase cDNA probe was kindly provided by Y. Kawakami (NCI,Bethesda, Md.). The probe for collagenase IV was 305 base pair PCRinsert of p16SPT19-1 (human 72 kDa type IV collagenase cDNA), subclonedinto the EcoRI/BamHI site of Bluescript SK-(2961 Base pairs). Theβ-actin probe (Oncor Inc., Gaithersburg, Md.), was used as control toensure equal loading of the samples.

Probes were labeled with ³² P dCTP (NEN) using a random primed DNAlabeling kit (Ready-To-Go, Pharmacia P-L-Biochemicals, UK). Membraneswere hybridized with probes (according to the Quikhyb protocol providedby Stratagene, La Jolla, Calif.) at 68° C. for 1 hour and washed twicefor 15 min each at room temperature with 2× standard saline-citrate/0.1×sodium dodecyl sulfate, and once at 60° C. for 30 min with 0.1× standardsaline-citrate/0.1× sodium dodcyl sulfate. Autoradiography was performedusing Kodak XAR5 films at -70° C. with intensifying screens.

Inhibition of Melanoma Cell Proliferation by NaPA and NaPB. Exposure ofhuman melanoma cells to NaPA or NaPB resulted in a dose-dependent growtharrest evident after three or more days of continued treatment (Table26). The decline in cell proliferation was accompanied by similarlyreduced DNA synthesis, but there was no change in cell viability.Compared to NaPA, NaPB was significantly more potent in inducingcytostasis, with IC₅₀ values ranging in seven of the eight tested celllines between 0.15 and 1.0 mM, versus 2.0-5.7 mM for NaPA. Reflecting onthe heterogenous response of melanoma cells, RPMI cells were moreresistant, requiring 8.4 mM NaPA or 1.5 mM NaPB to cause 50% inhibitionof growth. However, primary normal melanocytes were even less sensitive,with IC₅₀ of NaPA and NaPB being 11.0 mM and 2.8 mM, respectively.Phenylacetylglutamine (PAG), the end-metabolite of NaPB and NaPA inhumans, had no significant effect on tumor cell growth even at doses ashigh as 10 mM.

                  TABLE 26    ______________________________________    Growth Inhibition of Human Melanoma Cells by NaPA and NaPB                   IC.sub.50, mM    Cell Line        NaPA    NaPB    ______________________________________    624 mel          5.0     1.0    501 mel          2.0     0.15    888 mel          5.2     1.6    1011 mel         4.7     0.6    A375M            4.5     0.65    SKMEL 28         5.7     0.75    G361             4.2     N.D.    RPMI             8.4     1.5    ______________________________________     Data represents the mean number of cells in treated versus control     cultures, determined after 4 days of continuous exposure to drugs. Cell     viability was over 95% in cultures treated with 10 mM NaPA or 3 mM NaPB.

Alterations in Morphology and Pigmentation. In addition to affectingtumor cell proliferation, the aromatic fatty acids induced morphologicalalterations consistent with melanocyte differentiation, includingreduced nuclear to cytoplasm ratio, cell flattening with enhancedcytoskeletal organization, and in some cases, development of dendriticprocesses and increased melanogenesis. The degree of differentiationinduced by NaPB/NaPA varied significantly depending on the phenotype ofcells at the time treatment was initiated. For example, in A375cultures, which are composed of amenlanotic epithelial-like cells, onlymarginal morphological changes could be induced with occasionalappearance of dendritic cells, while dramatic alterations were observedin the more differentiated 1011 cultures Within 3-4 days of NaPBtreatment, 1011 cells appeared enlarged with a markedly increasedcytoplasm to nuclear ratio. Moreover, the cells had well organizedcytoskeletons (evidenced by staining for vimentin), developed longdendritic processes, and became highly melanotic (FIG. 40).Differentiation was progressive, involved the great majority of the cellpopulation, and became a stable trait after 2 weeks of continuoustreatment (see below). Consistent with its relative potency as acytostatic agent, NaPB was more effective than NaPA in promotingterminal differentiation of 1011 cells. Yet, neither drug was capable ofinducing major morphological changes nor melanin production in A375Mcultures, suggesting endogenous resistance to differentiation.

Reversibility of Antitumor Effects

The stability of NaPB-induced cytostasis and differentiation appeared todepend not only on the cells treated but also the duration of exposureto the drug. In 1011 cultures, the doubling time increased from 26±2 hrsto approximately 110 hrs following 4-day or longer treatment with 1.5 mMNaPB. If treatment was discontinued after one week, the doubling timewas reduced to 67 hr. However, if the duration was increased to 14 daysof continuous exposure to phenylbutyrate, the replication rate was notchanged significantly upon cessation of treatment (dell doubling every96 hrs). This contrasted the finding with A375M cells, which resumedgrowth in the absence of the drug (doubling time of 20-24 hrs). As shownin FIG. 40, 1011 mel cells exposed to 1.5 mM NaPB for 14 days had 31.1μg/ml melanin per 10⁶ cells; three days after treatment was discontinuedmelanin levels further increased to 39.9 μg/ml. The loss ofproliferative capacity, stable morphological change and persistentmelanogenesis in 1011 cells are all indicative of a terminaldifferentiation induced by NaPB.

Loss of Invasiveness and Tumorigenicity. Malignant melanoma are highlyinvasive and metastatic in vivo. Since biologically aggressive cellpopulations could still exhibit some differentiation markers, includingmelanogenesis, it was important to further examine the effect of NaPBand NaPA on the malignant phenotype. The ability of A375M, SKMEL 28 and1011 cells to degrade and cross tissue barriers was assessed by an invitro invasion assay using a modified Boyden chamber with amatrigel-coated filter. After 3-5 days of continuous treatment with thearomatic fatty acids there was a dose-dependent loss of invasivecapacity (Table 27). This in vitro indication of phenotypic reversioncorrelated with loss of tumorigenicity in vivo: A375M cells treated withNaPA for one week in culture, in contrast to untreated cells, failed toform tumors when transplanted s.c. into athymic mice (Table 27). Almostcomplete inhibition of invasiveness and tumorigenicity were observedwith 5 mM NaPA, a concentration that caused only partial cytostasis,indicating that malignant properties may be more vulnerable than cellproliferation in tissue culture dishes.

                  TABLE 27    ______________________________________    Reduced Invasiveness In vitro and Tumorigenicity in Athymic Mice            Invasion   Tumor Formation.sup.b    Treatment Through Matrigel.sup.a tumor diam.,    In vitro  (% of control)                           incidence mean (range).    ______________________________________    None      100          8/8       8.4                                     (2-20)    NaPA 2.5 mM                59 ± 5.5                           3/8       4.7                                     (0-12)    NaPA 5 mM 22.5 ± 4  1/8       2.0    NaPB 0.5 mM              44.1 ± 6.7                           N.D.      N.D.    NaPB 1.0 mM              16.1 ± 2.9                           N.D.      N.D.    ______________________________________     .sup.a A375 cells pretreated for 4 days were detached and assayed for     their ability to invade a reconstituted basement membrane using a matrige     Invasion Chamber. Comparable inhibition of invasiveness was documented     with SKMEL 28 and 1011 mel cells. Under the experimental conditions used,     2-3% of the untreated cells invaded the matrigel within 40 hrs (A375M,     1011) or 24 hrs (SKMEL 28).     .sup.b A375M cells were pretreated for one week with NaPA prior to being     injected (5 × 10.sup.5 cells/animal) s.c. into athymic mice. Result     indicate tumor incidence (tumor bearing/injected animals) and size of     tumors, as determined 4 weeks after cell transplantation.

Modulation of Gene Expression. The profound changes in tumor biologywere associated with alterations in expression of genes critical tomaintenance of the malignant phenotype, as well as those that couldaffect immunogeneicity in vivo. Specifically, cells treated with eitherNaPA or NaPB had reduced levels of collagenase type IV mRNA, coding fora 72,000 dalton metalloprotease. The latter is involved in degradationof extracellular stroma and basal lamina structures, with the potentialto facilitate tumor invasion and metastasis. As with other tumorresponses, the degree of changes in gene expression varied among thedifferent cells lines tested.

For example, while 5 mM NaPA completely abrogated collagenase IVexpression in SKMEL 28 cells, the specific transcript levels werereduced only 2 fold in A375M cells The decline in collagenase IV in A375treated with NaPA or NaPB was accompanied by increased expression of itsinhibitor TIMP II, suggesting that the net proteolytic activity and,consequently, invasiveness may be significantly reduced by the aromaticfatty acids.

Synthesis of the differentation marker melanin is regulated by theenzyme tyrosinase. Both NaPB and NaPA increased (albeit to a differentdegree) the steady-state levels of tyrosinase mRNA in 1011 cells, whichcorrelated with increased pigmentation. The Northern blot analysisindicated that the treated cells also had approximately 2 fold elevatedlevels of HLA-A3 mRNA. The results are consistent with previous findingsof increased MHC class I antigen expression in human leukemic andprostatic carcinoma cells following treatment with NaPA.

Potentiation of Activity of Other Antitumor Agents. Phenotypicheterogeneity is characteristic of tumor lesions in patients withmelanoma. The diversity in therapeutic responses of heterogeneous tumormasses would require appropriate combination treatment protocols. Datasummarized in FIG. 41 indicate that NaPA can significantly enhance theefficacy of other antitumor agents of clinical interest. Over 70%inhibition of melanoma cell proliferation was observed when NaPA wascombined with nontoxic, yet suboptimal concentrations of IFN-alpha,all-trans-RA, suramin, or 5AzadC.

Melanoma is becoming an increasingly important cause of disease anddeath worldwide, with estimated 70,000 new cases occuring each year.Despite major advances in cytotoxic chemotherapy, immunotherapy, orbiological therapy, the five year survival rate for individuals withdisseminated disease is 14%, and the median survival for patients withonly one site of metastasis is about seven months. In pursuit ofdeveloping new approaches to systemic therapy of advanced disease, theefficacy of the differentiation inducers phenylacetate andphenylbutyrate was explored. The two aromatic fatty acids have alreadybeen established as safe and effective in treatment of children andadults suffering from hyperammonemia (Brusilow, S. W., Danney, M.,Waber, L. J., Batshow, M., Burton, B., Levitsky, L., Roth, K.,McKeethren, C., Ward, J. (1984): Treatment of episodic hyperammonemia inchildren with inborn errors of urea synthesis. N. Eng. J. Med.310:1630-1634; Simell, O., Sipila, I., Rajantie, J., Valle, D. L.,Brusilow, S. W. (1986): Waste nitrogen excretion via amino acidacylation: benzoate and phenylacetate in lysinuric protein intolerance.Pediatric Res. 20:1117-1121), both have recently been approved by theFDA as investigative new drugs for the treatment of adults with cancer.Experimental data indicate that these relatively nontoxic compounds caninduce cytostasis and phenotypic reversion of human melanoma cells.

The degree of changes by NaPA and NaPB varied significantly among thetested cell lines, as would be expected considering the highlyheterogeneous nature of melanoma cells. There were however severalcommon alterations in tumor cell and molecular biology observed,including: (a) reduced proliferative capacity; (b) loss of invasivenessassociated with a decline in collagenase type IV and enhanced TIMP IIexpression; and, (c) increased expression of MHC class I antigens knownto affect tumor immunogeneicity in vivo. As with other differentiationinducers (Jardena Nordenberg, Lina Wasserman, Einat Beery, Doron Aloni,Hagit Malik, Kurt H. Stenzel, Abraham Novogrodsky (1986): Growthinhibition of murine melanoma by butyric acid and dimethylsulfoxide.Experimental Cell Research 162:77-85), the ability of cells to undergoterminal differentiation following exposure to NaPA/NaPB appeared todepend on their state of cell maturation at the time treatment wasinitiated. For example, in poorly differentiated A375 cells, the drugsinduced cytostasis with occasional appearance of dendritic cells, aslight increase in HLA expression, and loss of tumorgenicity in athymicmice; however, these cells remained nonpigmented andtyrosinase-negative, and resumed growth in culture once treatment ofcultures was discontinued. By contrast, exposure of the more mature 1011cells to the aromatic fatty acids resulted in profound and stablechanges in cell growth and morphology consistent with terminaldifferentiation. NaPB-treated 1011 cells became heavily pigmented andpolydendritic, and had elevated levels of tyrosinase and class I HLAmRNAs. Preliminary protein analysis indicates that NaPB causessignificant increase in both MHC class I and II surface antigens. Thesesurface proteins are necessary, if not sufficient, to evokeproliferative and cytotoxic T-cell responses against malignant melanomas(Guerry, D. IV, Alexander, M. A., Herlyn, M. F., Zehngebot, L. M.,Mitchell, K. F., Zmijewski, C. M., Lusk, E. J. (1984): HLA-DRhistocompatibility leukocyte antigens permit cultured human melanomacells from early but not advanced disease to stimulate autologouslymphocytes. J. Clin. Invest. 73:267-271; Fossati, G., Taramelli, D.,Balsari, A., Bogdanovich, J., Ferrone, S. (1984): Primary but notmetastatic human melanomas expressing DR antigens stimulate autologouslymphocytes. Int. J. Cancer 33:591-597).

NaPB and NaPA are closely related aromatic fatty acids. Phenylbutyrateis metabolized by mitochondrial β-oxidation to form phenylacetate.Phenylacetate, in turn, can be converted back to phenylbutyrate throughthe action of medium-chain fatty acid elongase. Reminiscent otherdifferentiation inducers such as retinoids and vitamin D derivatives,NaPA and NaPB were recently found to activate a member of the steroidnuclear receptor family, which functions as a transcriptional factor. Inaddition, both compounds can inhibit the mevalonate pathway ofcholesterol synthesis, and thus interfere with post-translationalprocessing of proteins critical to signal transduction and mitogenesis.At growth inhibitory concentrations, NaPB and NaPA also alter thepattern of DNA methylation, an epigenetic mechanism controlling thetranscription of various eukaryotic genes. NaPB's ability to activatethe expression of otherwise dormant methylation-dependent genes wasdocumented in experimental models and in man (Samid, D., Yeh, A.,Prasanna, P. (1992): Induction of erythroid differentiation and fetalhemoglobin production in human leukemic cells treated withphenylacetate. Blood 80:1576-1581; Brusilow, S. W., Danney, M., Waber,L. J., Batshow, M., Burton, B., Levitsky, L., Roth, K., McKeethren, C.,Ward, J. (1984): Treatment of episodic hyperammonemia in children withinborn errors of urea synthesis. N. Eng. J. Med. 310:1630-1634). Thus,NaPA and NaPB affect some common pathways leading to restored growthcontrol and cell maturation.

The findings with melanoma cells lines indicate, however, that NaPB is amore potent modulator of gene expression and cell biology compared toNaPA. The relative potency was confirmed in various hematopoietic andsolid tumors including lymphomas, gliomas, and adenocarcinomas of theprostate, breast, ovarian, lung and colon. The latter could be due tothe higher lipophilicity of NaPB, or be related to some differences inmechanisms of action. It is possible that, prior to being metabolized tophenylacetate, phenylbutyrate may act in an analogous way to the shortfatty acid, butyrate, a well characterized differentiation inducer withtherapeutic potential. There are, however, several differences in tumorresponses to phenylbutyrate versus butyrate. While the former increasedvimentin organization in human melanoma cells, butyrate was reported todecrease vimentin expression in B-16 melanoma cells (Ryan and Higgs1988). Furthermore, in contrast to NaPB, butyrate failed to induce, andin some cases even inhibited tyrosinase activity and melanization ofmelanoma cells (Jardena Nordenberg, Lina Wasserman, Einat Beery, DoronAloni, Hagit Malik, Kurt H. Stenzel, Abraham Novogrodsky (1986): Growthinhibition of murine melanoma by butyric acid and dimethylsulfoxide.Experimental Cell Research 162:77-85). Additional unique attributes ofphenylbutyrate with potential clinical implications include its extendedhalf-life (hours, versus minutes for butyrate), and the conversion tophenylacetate, itself a cytostatic and differentiation inducer. Beforeexcretion in the urine, phenylacetate must first be conjugated withglutamine to form PAG. High rates of urinary excretion of PAG associatedwith NaPA-related therapies, can beneficially deplete plasma glutamine(Simell, O., Sipila, I., Rajantie, J., Valle, D. L., Brusilow, S. W.(1986): Waste nitrogen excretion via amino acid acylation: benzoate andphenylacetate in lysinuric protein intolerance. Pediatric Res.20:1117-1121), the amino acid critical to tumor growth (HiroyukiTakahashi, Peter G. Parsons (1990): In vitro phenotypic alteration ofhuman melanoma cells induced by differentiating agents: Heterogeneouseffects on cellular growth and morphology, enzymatic activity, andantigenic expression. Pigment Cell Research 3:223-232).

It appears therefore that NaPB and NaPA cause reversion of malignantmelanoma cells and may be of value in management of this fatal disease.In developing these drugs for clinical use, it will be important toconsider the heterogeneity of melanomas. Metastatic melanoma cells varywidely in their growth rate, morphology and degree of pigmentations;such diversity was observed in different metastases and even among cellswithin a single lesion (Weber, G. (1983): Biochemical strategy of cancercells and the design of chemotherapy: G.H.A. Clowes Memorial Lecture.Cancer Res. 43:3466-3492; Hiroyuki Takahashi, Peter G. Parsons (1990):In vitro phenotypic alteration of human melanoma cells induced bydifferentiating agents: Heterogenous effects on cellular growth andmorphology, enzymatic activity, and antigenic expression. Pigment CellResearch 3:223-232). In view of the reversibility of effect seen withpoorly differentiated subpopulations, it is likely that prolongedduration of treatment would be required in order to benefit patients.Enhanced surface antigen expression and reduced production oftumor-secreted immunosuppression factors could eventually result intumor rejection by the host immune system. In cases where it might benecessary to target highly divergent tumor cell populations, thearomatic fatty acids could be combined with other antitumor agents toenhance efficacy, minimize adverse side effects, and prevent diseaserelapse. To this end, the potential of combining NaPA with otherdifferentiation inducers (retinoids), DNA hypomethylating drugs(5AzadC), growth factor antagonists (suramin), and cytokines(interferons) is demonstrated.

Both NaPA and NaPB are currently in clinical trials at the NCI. Ongoingphase I studies with NaPA, involving primarily patients with malignantglioma and hormone-refractory prostate cancer, indicate that therapeuticlevels can be achieved with no significant adverse effects and result inobjective clinical improvement in patient with advanced disease(described elsewhere herein).

SECTION O: NaPA and NaPB - Hydroxyurea Combination Treatment

Previous clinical trials have indicated that hydroxyurea could possesssome activity against prostate cancer. The in vitro activity ofhydroxyurea was evaluated in three hormone-refractory prostate cancercell lines, PC-3, DU-145, and PC-3M (as measured by the MTT method).Cytotoxicity was noted at concentrations ≧100 μM of hydroxyurea,requiring at least 120 hours of drug exposure (100 μM was theapproximate IC₅₀ for all three cell lines). Based on clinicalpharmacokinetic data, a dosing regimen was simulated to produce ahydroxyurea plasma concentration greater than 100 μM for 120 hours (1 gloading dose, followed by 500 mg every 6 hours for 5 days in a 70 kgman). Since this plasma concentration may result in an unacceptabledegree of myelosuppression, in vitro combinations studies were conductedwith hydroxyurea and phenylbutyrate, a new differentiating agent. Thesestudies resulted in a reduction of the hydroxyurea concentrationnecessary for a 50% growth inhibition (50 μM of hydroxyurea plus 0.5 mMof phenylbutyrate). A regimen designed to achieve that hydroxyureaconcentration (400 mg loading dose, followed by 200 mg every 6 hours for5 days) should be clinically achievable. The combination was furtherevaluated for use in treatment of patients with Stage D prostate cancer.

                  TABLE 30    ______________________________________    Clinical Trials Using Hydroxyurea in Prostate Cancer    Author Year   N     OR    SR    R   HU Dose    ______________________________________    Lerner 1977   30    63%   76%   N   80  mg/kg q3d × 6wks                        (19/30)                              (23/30)    Kvols  1977    5    60%   NR    N   3   gm/m.sup.2 q3d                        (3/5)    Loening           1981   28    14%   21%   Y   3   gm/m.sup.2 q3d                        (4/28)                               (6/28)    Mundy  1982   22    36%   68%   N   80  mg/kg q3d                        (8/22)                              (15/22)    Stephens           1984   69     4%   13%   Y   3.6 gm/m.sup.2 2d/wk                        .sup.  (9/69).sup.a    ______________________________________     N = number of patients receiving hydroxyurea     OR = objective response (stable, partial, complete)     SR = subjective response     R = randomized (N = No, Y = Yes)     HU Dose = Dose of hydroxyurea     .sup.a Stable response not reported, and only evaluated patients with sof     tissue disease     Response criteria varied between trials

Example 27

NaPA in combination with hydroxyurea-PC-3 and DU-145 cells

Two of the human prostate cell lines (PC-3 and DU-145) were obtainedfrom American Type Culture Collection, Rockville, Md. PC-3M was obtainedfrom James Kozlowski, M.D. at the University of Wisconsin via the NCICancer Treatment Screening Program at Frederick, Md. Hydroxyurea waspurchased from Sigma Chemical Co., St. Louis, Mo. Phenylbutyrate wasobtained from Elan Pharmaceutical Research Corporation (Gainesville,Ga.). Cell culture medium used was RPMI 1640 supplemented with 10% heatinactivated fetal bovine serum (FBS), penicillin 5,000 units/mL,streptomycin 5,000 μg/mL and 2 mM L-glutamine (Gibco Laboratories, GrandIsland, N.Y.).

The prostatic carcinoma cell lines were propagated in RPMI-1640, whichwas supplemented with 10% FBS and 1% antibiotics (penicillin andstreptomycin). The cells were grown to 80% confluent monolayers (6×10⁵cells/sq. cm) and cultivated in 75-sq. cm flasks (Nunc, Denmark). Cellswere harvested with trypsin (0.05%.):EDTA (0.02%) (Gibco Laboratories,Grand Island, N.Y.) solution and counted in a hemocytometer. The cellswere then seeded into 96 well microtiter plates (CoStar, Cambridge,Mass.) at a density of 3,000 cells per well in RPMI 1640 medium with 10%FBS and 1% penicillin-streptomycin solution and reincubated for 2.4hours to allow for cell reattachment (37° C., 5% CO₂ atmosphere).Hydroxyurea, diluted in tissue culture medium to yield a final wellvolume of 200 μL, was then added at specified concentrations (0.01, 0.1,1, 10, 100, 1,000, 10,000, and 100,000 μM) and left undisturbed for 120hours in the tissue culture incubator. Control cells were grown in anequal volume of medium. The3-(4,4-dimethyl-2-thiazolyl)-2,5-di-phenyl-2H-tetrazolium bromide (MTT,Sigma Chemical Co., St. Louis, Mo.) assay was used to estimate cellnumber (Beckloff G. L., Lerner H. J., Frost D, et al. Hydroxyurea(NSC-32065) in biologic fluids: dose-concentration relationship. CancerChemother Rep 1965;48:57-8). After exposure to hydroxyurea, MTT (20 μL,5 mg/mL in PBS) was added to the wells, incubated for 4 hours, andcentrifuged at 2,000 g, and the medium was decanted. Dimethyl sulfoxide(150 μL) was added to each well, the plates were shaken for 30 seconds,and the optical density at 540 nm was determined on a kinetic microplatereader (Bio-Tek EC340 Immunoplate-reader).

The synergistic activity studies were conducted with PC-3 cells byadding 0.5 mM and 1 mM of phenylbutyrate to 50 μM and 100 μM ofhydroxyurea. Similar in vitro conditions and durations of drug exposurewere utilized as described above. Cell numbers were determined byhemocytometer.

Statistical analysis was performed using the SAS computer program(version 6.02, SAS Institute, Inc., Cary, N.C.). The mean opticaldensity for the various concentrations of hydroxyurea versus controlwere compared by an adjusted two-sided Wilcoxon rank-sum test. An alphaof 0.05 was used to detect statistical difference. To determine therelationship between hydroxyurea dose and cell survival, the PC-3experiments were conducted five times for the PC-3 cell line and threetimes for both DU-145 and PC-3M. In each experiment there were six wellswere concentration. The experiments to determine the optimal durationwere performed once with 12 wells per fixed concentrations (five platesfor both methods).

For all three cell lines there was a dose-dependent decrease in thepercentage of cells surviving with hydroxyurea concentrations between 10and 1,000 μM (see FIG. 42. At a concentration of 100 μM there was anapproximate 50% reduction in cell survival (42.7%, 45.5%, and 52% forPC-3, DU-145, and PC-3M, respectively) for all three cell lines,compared to untreated cells (p=0.016). Increases in the hydroxyureaconcentration above 1,000 μM exerted little further effect on theinhibition of cell growth. (see FIG. 42). At concentrations less than100 μM, hydroxyurea was only cytostatic, but cytotoxicity increased withincreasing concentrations less than 100 μM, 86% of the total cells wereviable at 100 μM and 41.9% at 1,000 μM, determined by trypan bluestaining (Gibco Laboratories, Grand Island, N.Y.).

Two additional experiments were conducted with PC-3 to investigate therelationship between the duration of hydroxyurea exposure and inhibitionof cell growth. In the first experiment cell survival was assesseddaily, from 24 to 120 hours of exposure, with four differentconcentrations of hydroxyurea (0.1, 1.0, 10, and 100 μM); the MTT assaywas performed immediately upon completion of the specified duration ofhydroxyurea exposure. In the second experiment cells were exposed to thesame concentrations of hydroxyurea for varying periods of time (24, 48,72, 96 and 120 hours), but grown for a total of 120 hours beforeperforming the MTT assay. Drug exposure was terminated at the variousintervals by replacing drug-containing medium-with drug-free medium.This later experiment was performed to detect the possibility of arecovery in cell growth following brief periods of drug exposure. (seeFIG. 43). In both experiments there was decrease in the percentage ofsurviving cells with increasing duration of exposure. There was noevidence of a plateau in the effect by 120 hours, nor of a resurgence incell growth following drug washout.

With increasing concentrations of hydroxyurea there was a markedalteration in PC-3 cell morphology. Specifically, it appears that therewas a significant degree of cellular selectivity in the PC-3 cell linewhen exposed to 100 μM hydroxyurea concentrations compared to controlcells. The PC-3 cells are heterogenous and hydroxyurea may have selectedcells with a low nucleus/cytoplasm ratio (i.e. large flat cells).

The pharmacokinetic model and model parameters used to describe thedisposition of hydroxyurea in humans were derived from concentrationversus time data previously reported from clinical trials of this drug(Beckloff G. L., Lerner H. J., Frost D, et al. Hydroxyurea (NSC-32065)in biologic fluids: dose-concentration relationship. Cancer ChemotherRep 1965;48:57-8; Veale D. Cantwell B. M. J., Kerr N. et al. Phase 1study of high-dose hydroxyurea in lung cancer. Cancer ChemotherPharmacol 1988;21:53-6; Adamson R. H., Ague S. L., Hess S. M., et al.The distribution, excretion and metabolism of hydroxyurea-C14. J.Pharmacol and Exp Therapeut 1965;150:322-7; Belt R. J., Haas C. D.,Kennedy J, et al. Studies of hydroxyurea administered by continuousinfusion. Cancer 1980;46:455-62; Rosner F. Rubin H, Parise F. Studies onthe absorption, distribution, and excretion of hydroxyurea (NSC-32065).Cancer Cehmother Rep 1971;55:167-73; Creasey W. A., Capizzi R. L.,DeConti R. C. Clinical and biochemical studies of high-dose intermittentherapy of solid tumors with hydroxyurea (NSC-32065). Cancer ChemotherRep 1970;54:191-4; Bolton B. H., Woods L. A., Kaung D. T., et al. Asimple method of colorimeteric analysis for hydroxyurea (NSC-32065).Cancer Chemother Rep 1965;46:1-5; Davidson J. D., Winter T. S. A methodof analyzying for hydroxyurea in biological fluids. Cancer Chemother Rep1963;27:97-110). Curve-stripping techniques (Abbottbase™ PharmacokineticSystem, ver. 1.0) applied to the concentration versus time datafollowing a single oral dose of hydroxyurea (80 mg/kg) indicated thatthese data were best described by a single exponential decay, allowing asingle compartment open linear model to be used in predictinghydroxyurea's pharmacokinetics. The bioavailability of hydroxyurea hasnever been precisely determined but is reported to be "complete"(Donehower R. C. Hydroxyurea. In: Chabner B. A., Collins J. M., edsCancer Chemotherapy, Principles and Practice, Philadelphia: J. B.Lippincott 1990: 225-33) hence a bioavailability of 100% was assumed.Estimates of hydroxyurea's total body clearance were obtained using thetrapezoidal rule. Using the average parameter values derived from thismodel (volume of distribution, half-life, and clearance) the hydroxyureadosing regimen used by Lener et al. (80 mg/kg every third day; see FIG.44A) was simulated. This regimen provides only very brief exposure tohydroxyurea concentrations with in vitro antiproliferative activity. Aregimen of 1.0 g loading dose followed by 500 mg every 6 hours for 5days in a 70 kg man is shown in FIG. 44A; this regimen approximates theconditions required in vitro for 50% inhibition of prostate cancer cellgrowth.

Because hydroxyurea plasma concentrations of 100 μM or greater may causean unacceptable degree of myelosuppression in some patients, the abilityof phenylbutyrate to potentiate the efficacy of hydroxyurea wasexamined. Hydroxyurea at the suboptimal concentration of 50 μM wascombined with phenylbutyrate at concentrations of 0.5 mM and 1 mM. A 58%reduction in PC-3 cell growth was achieved when 0.5 mM of phenylbutyratewas added to 50 μM of hydroxyurea. As monotherapy, hydroxyurea (50 μM)reduced cell proliferation by only 38% and phenylbutryate, 0.5 mM and 1mM, by 38% and 55%, respectively. Approximately 80% inhibition of PC-3growth was observed when 50 μM hydroxyurea was combined with 1 mM ofphenylbutyrate, without affecting cell viability. (see FIG. 45).

The results of previous clinical trials indicate that hydroxyurea couldpossess some activity against hormone refractory prostate cancer. Leneret al. initially evaluated hydroxyurea in 1977 for the treatment stage Dprostate cancer (Lerner H. J., Malloy T. R. Hydroxyurea in stage Dcarcinoma of prostate. Urol 1977;10,35-8). The drug was administered asa single oral dose (80 mg/kg) every third day. A partial objective tumorresponse was reported in 15 of the 30 patients treated. Subsequent toLener's initial study there have been four additional clinical reportsof hydroxyurea in hormone-refractory prostate cancer (Kvols L. K., EaganR. T., Myers R. P. Evaluation of melphalan, ICRF-159, and hydroxyurea inmetastatic prostate cancer: a preliminary report. Cancer Treat Rep1977;61:311-2; Loening S. A., Scott W. W., deKernion J, et al. AComparison of hydroxyurea, methyl-chloroethyl-cyclohexy-nitrosourea andclylophosphamide in patients with advance carcinoma of the prostate. JUrol 1981;125:812-6; Mundy A. R. A pilot study of hydroxyurea in hormone"escaped" metastatic carcinoma of the prostate. Br J Urol 1982;54:20-5;Stephens R. L., Vaughn C, Lane M, et al. Adriamycin and cyclophosphamideversus hydroxyurea in advanced prostatic cancer. Cancer 1984;53:406-10).Mundy studies the same regimen of hydroxyurea administration (80 mg/kgevery third day) in 22 patients failing hormonal therapy and reportedimprovement in bone pain and performance status in 15 of these-patients(68%) within 6 weeks of starting therapy (Mundy A. R. A pilot study ofhydroxyurea in hormone "escaped" metastatic carcinoma of the prostate.Br J Urol 1982;54:20-5). Kvols et al. reported a single partialobjective response with hydroxyurea in a group of five patients (KvolsL. K., Eagan R. T., Myers R. P. Evaluation of melphalan, ICRF-159, andhydroxyurea in metastatic prostate cancer: a preliminary report. CancerTreat Rep 1977;61:311-2). The activity of hydroxyurea inhormone-refractory prostate cancer was evaluated in a randomized studycarried out by the National Prostatic Cancer Project. In this study 125patients failing hormonal therapy were randomly assigned to treatmentwith either methyl-chloroethyl-cyclohexy-nitrosurea (semustine),cyclophosphamide or hydroxyurea (Loening S. A., Scott W. W., deKernionJ, et al. A Comparison of hydroxyurea,methyl-chloroethyl-cyclohexy-nitrosourea and clylophosphamide inpatients with advance carcinoma of the prostate. J Urol 1981;125:812-6).Objective response was observed in 30% of the patients receivingsemustine, 35% of the patients receiving cyclophosphamide, and 15% ofthe patients receiving hydroxyurea. Nonetheless, improvement inperformance status was greater in the group of patients receivinghydroxyurea (18%, 5 of 28 patients) than in the other two groups (1 of27 patients for the sermustine group and 5 of 43 patients for thecyclophosphamide group), and it was equivalent in relieving bone pain.Another randomized, multicenter trial compared the regiment ofdoxorubicin plus cyclophosphamide to hydroxyurea in 158 patients withstage D prostate cancer (Stephens R. L., Vaughn C., Lane M., et al.Adriamycin and cyclophosphamide versus hydroxyurea in advanced prostaticcancer. Cancer 1984;53:406-10). No statistically significant differencein antitumor activity could be demonstrated between the two regimens(objective response rate 6 of 19 patients versus 1 of 24 patients,p=0.06). However, response was evaluated only in patients withmeasurable soft tissue disease, who represent a minority (15%) of thepatients with prostate cancer and whose biology may differ from that ofpatients having disease limited to bone (Hanks G. E., Myers C. E.,Scardino P. T. Cancer of the Prostate. In: DeVita V. T., Hellman S.,Rosenberg S. A., eds. Cancer: Principles and Practice of Oncology,Fourth Edition. Philadelphia: J. B. Lippincott 1993;1104).Unfortunately, comparing the response rates for these trials is impededby the use of heterogenous, imprecise and vague criteria at the time thetrials were conducted.

If the modes of antitumor activity observed in previous clinical trialsof hydroxyurea in hormone-refractory prostate cancer were the result ofthe drug's ability to inhibit DNA synthesis during S-phase, then it isclear that the dosing regimens used in those trials were suboptimal. Itis reasonable to assume that the regimen simulated in FIG. 44B wouldundoubtedly result in neutropenia. In order to reduce the toxic effectsof hydroxyurea, a reduced concentration was combined with a clinicallyachievable concentration of phenylbutyrate, a relatively non-toxicdifferentiation inducer currently undergoing clinical trials.Phenylbutyrate undergoes rapid conversion to phenylacetate in vivo byB-oxidation (Knoop F. Der Abbau aramatischer fettasaure Tierkorper.Beitr Chem Physiol Pathol 1905;6:150-62). Phenylbutyrate andphenylacetate have been used in children with urea cycle abnormalitiesand appear well tolerated in high-doses (Brusilow S. W., Horowich A. L.Urea cycle enzymes. In: Scriber C., Beaudet A. Sly W., ValleDr, eds.Metabolic Basis of inherited Diseases, New York: McGraw Hill 1987:629).Both agents have been recently identified as antineoplastic agentsaffecting tumor growth and maturation. In addition to causing selectivecytostasis, both induce malignant cells to undergo reversions to a morebenign phenotype. The data indicate that a combination of hydroxyureaand phenylbutyrate each used at cytostatic concentrations results insignificant inhibition of cell proliferation of the PC-3 cell cultures.

In conclusion, the results indicate that a much greater hydroxyureaexposure (i.e. >100 μM for at least 120 hours), as a single agent, isrequired for cytotoxic cell death than has been achieved in previousclinical trials. A regimen designed to achieve that concentration wouldmost likely result in unacceptable side effects. However, hydroxyurea incombination with phenylbutyrate has a clinical role in patients withhormone refractory metastatic prostate cancer. This combination regimenrequires a relatively low-dose of hydroxyurea (400 mg loading dosefollowed by 200 mg every 6 hours for 5 days, see FIG. 44C) to produce aconcentration of 50 μM, as well as a reduced concentration ofphenylbutyrate. The hydroxyurea dose employed may cause a mild reductionin neutrophils, but this reduction should be clinically tolerated. Theadverse effects associated with phenylbutyrate should not be additive(i.e., CNS depression) to the myelosuppression associated withhydroxyurea. Based on these results, this combination deserves furtherevaluation in patients with Stage D prostate cancer.

Section P: NaPA and NaPB Effects on Medulloblastoma & AstrocytomaDerived Cells

Medulloblastoma, and other malignant brain tumors, are heterogeneouswith regard to cell morphology, antigenic phenotype and biochemicalfeatures. A proportion of cells express a more differentiated phenotype.Malignant transformation of cells does not necessarily abrogate theirpotential for expression of differentiated characteristics includingcessation of proliferation (Sartorelli, A. C.: Malignant celldifferentiation as a potential therapeutic approach. Br. J. Cancer52:293-302, 1985; Marks P. A., Sheffery M., Rifkind R. A.: Induction oftransformed cells to terminal differentiation and the modulation of geneexpression. Cancer Res. 47:659-666, 1987). Spontaneous differentiationof medulloblastoma to a non-proliferative state occasionally does occur(DeChadarevian J.-P., Montes J. L., Govman A. M., Freman C. R.:Maturation of cerebellar neuroblastoma into ganglioneuroma withmelanosis. Cancer 59:69-76,1987), indicating that neuroectodermal tumorsmay be capable of differentiation to an effective signal. Thesebiological features and the current lack of sufficiently effectivetherapeutic strategies make agents which may induce differentiation ofthese tumors particularly interesting.

The mechanisms by which differentiating agents affect the growth andphenotypic characteristics of responsive cell lines have been studiedextensively and although their actions are incompletely understood, asubstantial body of evidence indicates that agents such as all-transretinoic acid (ATRA) interact with autocrine growth control pathways(Falk L.A., DeBenedetti F., Lohrey N. et al.: Induction of TGFβ1receptor expression and TGFβ1 protein production in retinoic acidtreated HL-60 cells. Blood 77:1248-1255, 1991). A variety of growthfactor molecules and their receptors are affected by exposure todifferentiating agents such as ATRA. In a number of neoplastic celllines as well as non-neoplastic cell types, ATRA induces production andsecretion of one or more isoforms of TGFβ (Falk L. A., DeBenedetti F.,Lohrey N. et al.: Induction of TGFβ1 receptor expression and TGFβ1protein production in retinoic acid treated HL-60 cells. Blood77:1248-1255, 1991).

The TGFβ autocrine growth regulatory pathways are of particular interestin primary central nervous system tumors. This pluripotential growthregulator is produced by both primary malignant astrocytoma tissue(Clark W. C., Bressler J.: TGFβ-like activity in tumors of the centralnervous system. J Neurosurg 68:920-924, 1988; Samuels V., Barett J. M.,Brochman S. et al.: Immunocytochemical study of transforming growthfactor expression by benign and malignant gliomas. Am J Pathol134:895-902, 1989) and by cell lines derived from such tumors (JenningsM. T., Macina R. J., Carver R. et al.: TGFβ1 and TGFβ2 are potentialgrowth regulators for low grade and malignant gliomas in vitro withevidence in support of an autocrine hypothesis. Int. J. Cancer49:129-139, 1991). The immunosuppressive effects of malignantastrocytoma cells on cocultured lymphocytes in vitro has beenconvincingly linked to TGFβ production and can be partially neutralizedby antibodies against TGFβ. TGFβ has been shown to be a growth regulatorfor gliomas in vitro (Jennings M. T., Macina R. J., Carver R. et al.:TGFβ1 and TGFβ2 are potential growth regulators for low grade andmalignant gliomas in vitro with evidence in support of an autocrinehypothesis. Int. J. Cancer 49:129-139, 1991). The role of the TGFβpathway in growth regulation of medulloblastoma is less well establishedthan for malignant astrocytomas. The antiproliferative effect of ATRA onDaoy medulloblastoma cells is associated with increased secretion ofTGFβ2 and with induction of TGFβ receptor expression. Because the TGFβfamily of growth factors plays such an important role in the biology ofmalignant astrocytomas, the initial focus has been on this potentialautocrine pathway.

The effects of a nontoxic differentiation inducer, phenylacetate (PA),on neuroectodermal tumor-derived cell lines were examined. Treatment ofmedulloblastoma (Daoy, D283) and glioma (U251, C6, RG2) cell linesresulted in a dose-dependent decline in DNA synthesis and cellproliferation, with ID₅₀ values ranging from 6.3 to 14.6 mM. PAincreased TGFβ2 production by medulloblastoma Daoy cells; however,neutralizing antibodies against either TFGβ2 or TGFβ1 failed to blockthe growth arrest observed, suggesting that, unlike otherdifferentiation agents, such as retinoic acid, the antiproliferativeeffect of PA is not mediated by a TGFβ pathway. In addition tocytostasis, PA induced marked morphological changes in U251 and C6glioma cells associated with increased abundance of GFAP-positiveprocesses. Although the morphology of PA-treated medulloblastoma cellswas not significantly altered, the D283 cells exhibited increasedexpression of neurofilament proteins and Hu antigen, indicative ofdifferentiation along a neuronal pathway. The effects of PA on themedulloblastoma cell lines were compared to the effects of PA on thewell established human neuroblastoma differentiation model BE(2)C, whichis capable of a bidirectional differentiation towards a neuronal or aglial/schwann cell pathway. In BE(2)C cells PA induced differentiationtowards a schwann/glial cell like phenotype, suggesting that the choiceof differentiation pathway is cell type and agent specific.

Example 28

NaPA Efficacy on Astrocytoma Cells

Cell culturing techniques. Human glioma (U251), rat glioma (C6, RG2),human medulloblastoma (Daoy, D283), human neuroblastoma (BE(2)C) (allfrom ATCC) and the murine lung fibroblast cell line MuLv1 (provided byDr. S. Cheifietz) were routinely maintained in Eagles minimal essentialmedia (MEM) (GIBCO) supplemented with 10% fetal calf serum (FCS),nonessential amino acids (NEAA), L-glutamine (2 mM), penicillin (100U/ml) and streptomycin (100 ug/ml) (all medium constituents werepurchased from Gibco, Grand Island, N.Y., USA). Adherent cell lines wereplated in T-150 tissue culture flasks (Becton-Dickenson) and passagedusing trypsinization when 70% confluent. The cell line growing insuspension (D283) was maintained at a cell density of 10⁵ -10⁶ cells perml. Cells were grown at 37° C. In a humidified 5% CO₂ incubator. Forglutamine depletion studies U251 cells were adapted to serum- andglutamine-free Ultraculture medium (Hyclone). For proliferation studieswith exogenous TGFβ1 and TGFβ2 and neutralizing antibodies against TGFβDaoy cells were grown in MEM containing 0.2% FCS and U251 cells werecultured in serum-free Ultraculture medium (Hyclone).

PA- and ATRA-treatment of cell lines. Phenylacetic acid (Sigma) wasdissolved in MEM to make a 100 mM stock solution and the pH was adjustedto 7.2 with NaOH. Then the MEM was supplemented with 10%heat-inactivated FCS. All-trans-retinoic acid (ATRA) was dissolved inethanol to prepare a 10⁻³ M stock solution and added to the media toyield final concentrations ranging from 10⁻⁷ to 10⁻⁶ M.

Cell proliferation assay. For proliferation studies tumor cells in logphase growth were harvested and resuspended at a concentration of 20,000cells/ml; 100 ul/well of the cell suspension was placed into 96-wellmicrotiter plates (Costar). After 24 h the cells were incubated with anequal volume of the serial dilutions of PA to yield final concentrationsranging from 5 to 20 mM PA. MEM containing 10% FCS served as a control.Cell proliferation was determined at 24 h intervals by ³ H-thymidineincorporation. One μCi of ³ H-thymidine (Amersham) was added to eachwell and the microtiter plates were incubated for 4 h at 37° C. Then thecells were harvested onto glass fiber filters using a semi-automatedcell harvester (Cambridge Biotechnologies) and ³ H-thymidine activitywas quantitated using a liquid scintillation counter (Hewlett Packard).All experiments were set up in quladruplicates.

Effects of exogenous TGFβ. To determine the effects of exogenous TGFβ onDaoy cells recombinant human TGFβ1 and TGFβ2 (R&D Systems) atconcentrations ranging from 0.5 PM to 1 nM were added to similarlyprepared microtiter plates for 24 h prior to determining ³ H-thymidineuptake.

Neutralizing studies with anti-human TGFβ1 and TGFβ2 antibodies.Polyclonal antibodies against human TGFβ1 and TGFβ2 (R&D Systems) werecoincubated in concentrations of 10 to 40 μg/ml with Daoy cells grown in0.2% FCS or U251 cells grown in serum-free medium (Ultraculture,Hyclone) exposed to PA 10 mM. Proliferation was determined by ³H-thymidine incorporation as described above.

Washout studies. Cells were seeded similar to proliferation studies asdescribed before except for a number of 250 cells per well. Two daysafter treatment with PA at various concentrations the agent containingmedium was replaced by plain culture medium and ³ H-thymidine uptake wasmeasured after 4, 8, 24, 48 and 72 h.

Immunocytochemistry. Adherent cell lines were grown on Lab-Tek 8-chamberslides (Nunc) under the same conditions previously described for cellcultures. After 24 h the cells were treated with PA 20, 10 or 5 mm for 4to 7 days. After fixation in cold acetone immunoreactivity wasdetermined using the avidin-biotin-peroxidase complex method of Hsu andcolleagues (Hus S. M., Raine L. and Fanger H.: Use ofavidin-biotin-peroxidase techniques: a comparison between ABC andunlabeled antibody (PAP) procedures. J. Histochem Cytochem 29:577-580,1981). For nonadherent cell lines, cytocentrifuged preparations weremade using a Cytospin II (Shandon) and the cells fixed essentially asdescribed above.

Mouse mAbs specific against GFAP (clone G-A-5), NF triplet proteins (68kD (clone NR4), 160 kD (clone BF10) and 200 kD (clone RT97)),synaptophysin (clone SY38) or vimentin (clone V9) were purchased fromBoehringer-Mannheim. Anti-TGFalpha (clone 213-4.4) and EGF-receptor(clone) antibodies were obtained from Oncogene Science. Anti-HLA class I(clone ) and II (clone L243) mAbs were purchased from Becton-Dickenson.Irrelevant mouse antibodies (MOPC-21, Sigma) served as a negativecontrol.

Anti-Hu IgG (provided by Dr. J. B. Posner) was prepared from a hightiter anti-Hu serum and conjugated to biotin as reported previously(Szabo A., Dalmau J., Manley G., Rosenfeld M., Wong E., Henson J.,Posner J. B., Furneaux H.: HuD, a paraneoplastic encephalomyelitisantigen, contains RNA-binding domains and is homologous to Elav andSex-lethal. Cell 67:325-333, 1991). Frozen sections (7 μm thick) ofhuman brain served as a positive control. Incubation with biotinylatedIgG from a normal individual was taken as negative control.

Western blotting. Tumor cells were solubilized in 50 mM Tris-HCl, pH7.2, containing 0.5% NP-40 and proteinase inhibitors(phenylmethylsulfonylfluoride, pepstatin, leupeptin, soybean trypsininhibitor, aprotinin and benzamidine). Equal aliquots (15-30 μg) of theprotein extracts were electrophoresed in 8 or 10% SDS-PAGE gels underdenaturing conditions. The proteins were then electroblotted (HoefflerTransblot system) from the gel onto 0.2 μm poresized nitrocellulose.Blots were blocked in 5% milk for 2 h, incubated with the primaryantibody (mAb in mouse or human serum) for 1 h and detected usingHRP-conjugated sheep anti-IgG (Amersham) directed against theappropriate species of primary antibody and the chemiluminescenttechnique (Amersham).

For Western analysis of Hu a high titer serum of a patient with anti-Husyndrome was used and serum from a healthy individual served as anegative control.

Cell cycle analysis. For cell cycle studies, Daoy cells were plated inT-150 culture flasks in serum-containing medium. After 24 h ofincubation, the culture medium was replaced by serum-free medium(Ultraculture, Hyclone) for 24 h to synchronize the cell cultures; thenthe cultures were refed medium containing PA 10 mM or plain serumcontaining medium. Cells were harvested after 48, stained with acridineorange and analyzed on a FAC-Scan (Becton-Dickenson).

RT-PCR assay of HuD-expression. Total RNA (1 μg) from tumor cell lineswas reverse transcripted with random hexamers (2.5 μM) for 50 min at 37°C. In a reaction volume of 20 μl containing the following: 1 mM eachdNTP, 1 U/μl RNase inhibitor, and 2.5 U/μl MMLV-RT (Gibco). RT reactionswere terminated by incubation at 99° C. for 5 min. One-twentieth of theRT reaction product was used for PCR amplification. PCR was carried outin a 1×PCR buffer (50 mM Tris pH 9.5, 1.5 mM MgCl₂, 20 mM ammoniumsulfate), 0.2 mM each dNTP, 5 μCi of (³² P)dCTP, 0.5 μM each upstreamand downstream primer (CCAGGCCCTGCTCTCCC, AGGCTTGTCATTCCATC) for HuD andfor β2-microglobulin, and 1 U of Taq polymerase (Perkin-Elmer Cetus).PCR analysis was performed in a Perkin-Elmer Cetus DNA cycler with thefollowing temperature profile: 1 min at 90° C.; 35 cycles of 30 s at 55°C., 1 min at 72° C., and 1 min at 92° C.; 2 min at 55° C.; and 10 min at72° C. One-tenth of the PCR product was electrophoresed in 6% acrylamidegel, and the PCR product was analyzed by autoradiography.

TGFβ bioassay. Conditioned media was produced by incubating Daoy cellsin the presence of PA 10 mM in T-150 culture flasks. The culture mediawas aspirated and concentrated 20 fold to a final volume of 200 μl usingAmicon centrifugal concentrators. The concentrated media was transferredto silicon treated polypropylene microfuge tubes (Danville Scientific)and protease inhibitors (same as mentioned before) added. To activatelatent TGFβ, the samples were acidified using acetic acid andsubsequently neutralized with 5N NaOH prior to the assay.

The MuLv1 cells were harvested by scraping flasks, washed in serum freeMEM, resuspended in MEM+0.2% FCS and seeded into 96 well microtiterplates at a cell density of 2000 cells per well in 50 μl aliquots. Afterthe plates were incubated for 4 h, 150 μl of conditioned media from celllines to be tested for TGFβ production was added per well. The plateswere incubated for 24 h and then cell proliferation was measured by ³H-thymidine incorporation as described previously. A standard curverelating TGFβ concentration to rate of ³ H-thymidine incorporation wasgenerated using human recombinant TGFβ1 (R&D systems), ranging from 0.1pM to 10 nM.

Elisa for TGFβ2. Conditioned media was concentrated 20 fold using Amiconconcentrators to a final volume of 200 μl. All the concentration stepswere performed at 4° C. Protease inhibitors (same as listed for Westernanalysis) were added to the concentrated samples prior to storage at-70° C. Latent TGFβ was activated by adding glacial acetic acid to thesamples to yield a final concentration of 1M acetic acid. Then thesamples were evaporated to dryness using a Speed-Vac and redissolved in200 μl of distilled water. The reconstitution/drying step was repeatedfor a total of 3 washes. Then the samples were redissolved in the sampletreatment buffer RD5B provided in the kit and the capture ELISA protocolperformed following the manufacturer's instructions. All data pointswere performed in duplicate. The colorimetric reaction was quantitatedby measuring absorbance at 450 nm using a Bio-RAD Microplate reader andthe Biorad Elisamatic software. A standard curve was generated usinghuman recombinant TGFβ2 as the protein standard.

Effects of PA on cell growth and cell cycle. All 6 cell lines showed adose-dependent growth inhibition after incubation with PA (FIG. 46) witha maximum effect after 48 to 96 h. The human malignant glioma cell lineU251, which was previously reported to be ATRA resistant (Yung W. K. A.,Lotan R., Lee P., Lotan D., Steck P. A.: Modulation of growth andepidermal growth factor receptor activity by retinoic acid in humanglioma cells. Cancer Res 49:1014-1019, 1989), also responded to PA. Thehuman tumor cell lines as well as the rat glioma cell line C6 showedID₅₀ values for PA between 5.8 and 9.5 mM, whereas the rat glioma cellline RG2 appeared to be less sensitive with an ID₅₀ of 14.6 mM (Table28). Trypan blue exclusion demonstrated greater than 90% viability of PAtreated cell cultures. When PA was removed after 48 h a clear washouteffect was noted in 3 cell lines studied (Daoy, U251 and C6).

                  TABLE 28    ______________________________________    ID.sub.50 Determinations for PA induced growth    inhibition of various tumor cell lines    Cell Line       ID.sub.50  mM!    ______________________________________    D-283 MED       9    Daoy MED        6.3    U-251 MG        8.2    RG-2 rat glioma 14.6    C6 rat glioma   8.5    BE(2)C          9.5    ______________________________________

Cell cycle analysis of Daoy cells treated with 10 mM PA for 2 daysshowed an accumulation of cells in G0/G1 with an equivalent reduction inthe percentage of cells in the S- and G2/M-phase. Prior to treatment34.2% of Daoy cells were in G0/G1, 55.5% in S, and 10.3% in G2/M phase.After treatment with PA 51.9% were in G1, 39.9% in S, and 8.2% in G2/Mphase.

Effects of PA on morphology and phenotype of glioma cell lines. PAinduced marked morphological changes in U251 cells. Prior to treatment,the cells were characterized by spindly bipolar GFAP positive processes.After PA treatment for 4 days, cytoplasmic processes were more extensiveand complex. The cytoplasmic/nuclear ratio was increased greatly withenlargement of the cell body and elaboration of a radiating stellatearray of cytoplasmic processes resembling those of hyperplasticastrocytes. The extent of these morphological changes were dose-related.Similar effects were seen in C6 rat glioma cells. Most untreated cellswere GFAP negative, and in the few positive cells the immunoreactivitywas localized in the perinuclear region. Although Western analysis forGFAP expression in both cell lines failed to show a clearcutquantitative change after exposure to PA, the fraction of cells stainingfor GFAP increased and filamentous immunoreactivity appeared in thecellular processes. Untreated U251 cells also expressed vimentin, EGFr,TGFalpha and ELA class I antigens. None of these cellular markers wasmodified by PA treatment. After discontinuation of PA treatment after 4days all morphological changes of U251 cells reverted to thepretreatment state within 3 to 5 days.

Effects of PA on the differentiation state of medulloblastoma celllines. In short term cultures of the medulloblastoma cell lines Daoy andD283 growth inhibition by PA was not accompanied by gross morphologicalchanges. Daoy cells expressed both class I and class II HLAimmunoreactivity; these phenotypic markers remained unchanged after PA(10 mM) treatment. Neither of the 2 cell lines expressed synaptophysinand GFAP before or after treatment with PA. Untreated Daoy cells werereactive for EGFr and TGFα; immunohistochemical staining for EGFr andTGFα was not affected by PA treatment.

Expression of Hu is one of the earliest markers of neuronal phenotype inthe peripheral nervous system of the chick (See Marusitch) and one ofthe earliest markers of neuronal phenotype in the developing centralnervous system of the mouse. Previous studies of the expression of Hu inhuman normal tissue and tumor tissue demonstrated that the Hu antigen ishighly restricted to the nervous system, small cell lung cancer tumorsand neuroblastomas (Dalmau J., Furneaux H. M., Cordon-Cardo C., PosnerJ. B.: The expression of the Hu (paraneoplasticencephalomyelitis/sensory neuronopathy) antigen in human normal andtumor tissues. Am J Pathol 141:881-886, 1992). Immunocytochemistry usingbiotinylated anti-Hu antisera showed Hu antigen expression in D283 andBE(2)C cells, whereas Daoy cells and all glioma cell lines werenegative. Results of immunostaining were confirmed by Western analysisand RT-PCR.

For Western analysis, cells were solubilized in 50 mM Tris buffercontaining 0.5% NP-40 and oiled for 7 min in the presence of2-mercaptoethanol. Proteins were separated on 10% SDS-PAGE gels with 20ug of protein loaded per lane. Proteins were transferred from the gel tonitrocellulose by electroblotting. Immunoreactivity was determined byexposing the blots to a human serum with high titer anti-Hu antibodiesor serum from a normal donor and then incubated with HRP-conjugatedsheep-anti human IgG. The blots were then autoradiographed using theenhanced chemiluminescent system (Amersham). Hu-fusion protein andprotein extracts of cortical neurons served as positive and normal humanserum as a negative control. For RT-PCR analysis 1 μg of total RNA fromtumor cell lines was reverse transcripted with random hexamers andone-twentieth of the RT reaction product was used for PCR amplificationwith HuD and β2-microglobulin specific primers. The PCR product waselectrophoresed in a 6% acrylamide gel and analyzed by autoradiography.

The Hu antigen was detectable as a band with a molecular weight of 35 to40 kDa on blots of denaturing SDS-PAGE gels. None of the cell linesreacted with normal human serum.

Daoy cells were negative for NFs whereas D283 cells expressed all 3 NFproteins, determined immunocytochemically. By immunocytochemistryquantitative changes after treatment with PA were seen for NF-M and Huexpression. After exposure to PA (10 mM) for 7 days NF-M and Huimmunoreactivity were enhanced. Western analysis of untreated and PAtreated D283 cell homogenates confirmed these quantitative changes inNF-M and Hu protein expression. Western analysis was performed for Huand NF-M expression of untreated and PA (10 mM, 7 d) treated D283 cellslysed in 50 mM Tris containing 0.5% NP-40 and separated under denaturingconditions by electrophoresis through 10% SDS-PAGE gels and transferredto nitrocellulose paper by electroblotting. Specific immunoreactivitywas determined using human serum containing high titer anti-Huantibodies and murine anti-NF-M respectively and the enhancedchemiluminescent technique (Amersham). Normal human serum or theappropriate isotype matched irrelevant mAb served as control.Immunostaining for NF-L revealed similar but less obvious changes afterPA treatment and NF-H remained unchanged.

Effects of PA on the human neuroblastoma differentiation model BE(2)C.The effects of PA on medulloblastoma cell lines were compared to thehuman neuroblastoma cell line BE(2)C, which represents a wellestablished bipotential differentiation model for neuronal andglial/schwann cell differentiation, indicated by changes in phenotypicmarkers, such as NF proteins, Hu and Vimentin, with ATRA inducing aneuronal and BUdR a glial/schwann cell phenotype (Biedler J. L., CasalsD., Chang T., Meyers M. B., Spengler B. A., Ross R. A.: Multidrugresistant human neuroblastoma cells are more differentiated thancontrols and retinoic acid further induces lineage-specificdifferentiation. Advances in Neuroblastoma Research 3:181-191, 1991,Wiley-Liss, Inc.; Ross R. A., Bossart E., Spengler B. A., Biedler J. L.:Multipotent capacity of morphologically intermediate (I-type) humanneuroblastoma cells after treatment with differentiation-inducing drugs.Advances in Neuroblastoma Research 3:193-201, 1991, Wiley-Liss, Inc.;Ciccarone V., Spengler B. A., Meyers M. B., Biedler J. L., Ross R. A.:Phenotypic diversification in human neuroblastoma cells: Expression ofneural crest lineages. Cancer Res 49:219-225, 1989; Ross Ra, Spengler B.A., Rettig W. R., Biedler J. L.: Permanent phenotypic conversion ofhuman neuroblastoma I-type cells. Proc Am Assoc Cancer Res 35:44, 1993).BE(2)C neuroblastoma cells showed a decrease in Hu and NF-M proteinexpression and an increase in vimentin expression following PA treatment(10 mM) for 7 days by both immunocytochemistry and Western analysis.Cells were lysed using 50 mM Tris containing 0.5% NP-40 and then theproteins were separated under denaturing conditions by electrophoresisthrough 8-10% SDS-PAGE gels and transferred to nitrocellulose paper byelectroblotting. Specific immunoreactivity was determined using murineanti-vimentin mAb, human serum containing high titer anti-Hu antibodies,and murine anti-NF-M respectively and the enhanced chemiluminescenttechnique (Amersham). Normal human serum or the appropriate isotypematched irrelevant mAb served as control.

Role of TGFβ in PA-mediated growth inhibition. To test the hypothesisthat TGFβ is involved in mediating the growth inhibitory effects of PAconditioned media of Daoy cells was studied before and after treatmentwith PA. Incubation of Daoy cells with PA 10 mM led to increased amountsof TGFβ in the conditioned media determined by the mink lung cell(MuLv1) bioassay. After 4 days of incubation, the control mediacontained 0.5 pM and the PA treated conditioned media contained 3.0 pMTGFβ. Further characterization of the secreted bioactive TGFβ proteinusing an ELISA assay specific for TGFβ2 indicated that the secretion ofTGFβ2 was stimulated by PA.

Addition of exogenous human recombinant TGFβ1 or TGFβ2 at concentrationsranging form 0.5 pM to 1 nM did not inhibit growth of untreated Daoycells maintained in MEM containing 0.2% FCS. These TGFβ proteinsinhibited the growth of MuLv1 cells profoundly after a 24 h exposurewith an ID₅₀ of 2 pM. Blocking studies with neutralizing antibodiesagainst TGFβ1 and TGFβ2, at concentrations up to 20 μg/ml, had noeffects on PA induced inhibition on Daoy and U251 cell growth (FIG. 47).However, these concentrations of antibodies were capable of neutralizingthe growth inhibitory effects of exogenous TGFβ1 and TGFβ2 on MuLv1cells.

As described herein and in the copending application, PA can inducedifferentiation of various hematological malignancies as well as solidtumors. These in vitro effects on human cell lines were noted atconcentrations that have been achieved without toxicity in childrentreated with PA for urea cycle disorders. The results demonstrate thatPA is a growth inhibitor for glioma and medulloblastoma-derived celllines. PA also showed antiproliferative effects in the U251 glioma cellline, which was previously reported to be resistant to ATRA (Yung W. K.A., Lotan R., Lee P., Lotan D., Steck P. A.: Modulation of growth andepidermal growth factor receptor activity by retinoic acid in humanglioma cells. Cancer Res 49:1014-1019, 1989). Cell cycle analysisperformed in Daoy cells showed an accumulation of cells in the G0/G1phase, which was similar to ATRA-induced effects on cell cyclepreviously reported for glioma cell lines (Rutka J. T., DeArmond S. J.,Giblin J., McCullock J. R., Wilson C. B., Rosenblum M. L.: Effect ofretinoids on the proliferation, morphology and expression of glialfibrillary acidic protein of an anaplastic astrocytoma cell line. Int JCancer 42:419-427, 1988). The rapid onset of growth inhibition is notdue to cytotoxicity, since cells resume proliferation and revert totheir previous morphology after washout of PA.

PA induced morphological and immunocytochemical changes consistent withastrocytic differentiation in glioma cell lines. Both glioma cell linesstudied showed marked morphological changes after PA treatment withincreased cytoplasmic/nuclear ratio and the formation of GFAP positiveprocesses. GFAP immunoreactivity appeared more intense in these newlyformed processes. However, Western analysis failed to demonstrate aclear quantitative change in GFAP expression after PA treatment.Previous studies of differentiation agents in glioma cell lines showedsimilar findings on GFAP expression in U251 and C6 cells after treatmentwith mycophenolic acid (Lipsky R. H., Siverman S. J.: Effects ofmycophenolic acid on detection of glial filaments in human and ratastrocytoma cultures. Cancer Res 47:4900-4904, 1987). The enhancedimmunoreactivity for GFAP without increase in GFAP levels might be dueto a modification of existing proteins by affecting the organization ofassembled GFAP filaments. The discrepancy between immunoblotting andimmunocytochemistry may reflect the inability of the blotting techniqueto detect changes occurring in a subpopulation of the cells in aheterogenous population. ATRA induced phenotypic changes in responsiveglioma cell lines which are similar to those produced by PA (Yung W. K.A., Lotan R., Lee P., Lotan D., Steck P. A.: Modulation of growth andepidermal growth factor receptor activity by retinoic acid in humanglioma cells. Cancer Res 49:1014-1019, 1989). Human glioma cell lineswere found to be more sensitive to ATRA compared to rodent cell lines.Interestingly, the least PA sensitive cell line was also of rodentorigin.

In short term cultures (up to 7 days) of both medulloblastoma cell linesthe growth inhibitory effects of PA were not associated withmorphological changes and the Daoy cells showed no changes in cellularexpression of the numerous markers tested, including NF proteins,synaptophysin, Hu, TGFalpha, EGFr, GFAP and HLA class I and II. Thistemporal dissociation of antiproliferative effects from phenotypicchanges has been reported for other differentiation agents (Ross Ra,Spengler B. A., Rettig W. R., Biedler J. L.: Permanent phenotypicconversion of human neuroblastoma I-type cells. Proc Am Assoc Cancer Res35:44, 1993; Pleasure S. J., Page C., Lee V. M.-Y.: Pure, postmitotic,polarized human neurons derived from NTera2 cells provide a system forexpressing exogenous proteins in terminally differentiated neurons. TheJ of Neuroscience 12:1803-1815, 1992). Daoy cells also revealed nomorphological changes after treatment with dBcAMP (Jacobsen P. F.,Jenkyn D. J., Papadimitriou J. M.: Establishment of a humanmedulloblastoma cell line and its heterotransplantation into nude mice.J. Neuropath Exp Neurol 44:472-495, 1985). ATRA treatment showedprofound growth inhibitory effects in medulloblastoma cell lines withoutdemonstrable concurrent morphologic changes (Agrawil A., Martell L. A.,Ross D. A., Muraszko K. M.: Reinoic acid modulation of proliferation anddifferentiation in brain tumors. Proc Am Assoc Cancer Res 34:20, 1993(abstract)), which is in agreement with data on ATRA studies inmedulloblastoma cell lines (Lieberman F., Finzi D., Ferro J.: Retinoicacid decreases proliferation and Hu expression in medulloblastoma cells.Ann Neurol 32:286, 1992).

Conflicting reports about the clinical relevance of glial or neuronaldifferentiation in medulloblastoma as indicated by GFAP and NF proteinexpression and morphologic criteria (Schofield D.: Diagnostichistopathology, cytogenetics, and molecular markers of pediatric braintumors. Neurosurg Clin N Amer 3:723-738, 1992) reflect an incompleteunderstanding of the biochemical changes correlated with growthinhibition. In addition, the cell of origin still remains unclear.Phenotypic characterization of medulloblastoma cell lines suggests atleast 2 distinguishable phenotypic patterns, with relative differencesin expression of neuronal- or glial-associated antigens, with D283representing a neuronal and Daoy a nonneuronal phenotype ofmedulloblastoma cell lines (He X., Skapek S. X., Wikstrand C. J.,Friedman H. S., Trojanowski J. Q., Kemeshead J. T., Coakham H. B.,Bigner S. H. and Bigner D. D.: Phenotypic analysis of four humanmedulloblastoma cell lines and transplantable xenografts. J NeuropatholExper Neurol 48:48-68, 1989).

NF protein expression has been used to identify neuronal differentiation(Tremblay F. G., Lee M.-Y., Trojanowski J. Q.: Expression of vimentin,glial filament and neurofilament proteins in primitive childhood braintumors. Acta Neuropathol (Berl) 68:209-244, 1985). In mature,post-mitotic neurons, NF proteins consist of 3 constituent proteins,termed light, medium and heavy based on different molecular weights indenaturing SDS gels. A previously proposed ontogenic schema suggeststhat the appearance of NF-H correlates with the post-mitotic state(Molenaar W. M., Jansson D., Goild V. E. et al.: Molecular markers ofpediatric neuroectodermal tumors and other pediatric nervous systemtumors. lab Invest 61:635-643, 1989). Previous studies of NF-expressionin medulloblastoma cell lines (Baker D. L., Reddy U. R., Pleasure S.,Hardy M., Williams M., Tartaglione M., Biegel J. A., Emanuel B. S.,Presti P. L., Kreidler B., Trojanowski J. Q., Evans A., Roy A. R.,Venkataakrishnan G., Chen J., Ross A. H., Pleasure D.: Human centralnervous system primitive neuroectodermal tumor expressing NGF receptors:CHP707m. Ann Neurol 28:136-145, 1990) have demonstrated that the CHP707mPNET line expresses only NF-L suggesting that this cell line reflects animmature state relative to D283 cells. The D283 cell line expressed all3 forms, although in an abnormal way (Trojanowski J. A., Friedman H. S.,Burger P. C., Bigner D. D.: A rapidly dividing human medulloblastomacell line (D283) expresses all 3 neurofilament subunits. Am J Pathol126:358-363, 1987), suggesting this defect indicates aberrant NF proteinconstruction and disordered intermediate filament function, which may bea consequence of neoplastic transformation (He X., Skapek S. X.,Wikstrand C. J., Friedman H. S., Trojanowski J. Q., Kemeshead J. T.,Coakham H. B., Bigner S. H. and Bigner D. D.: Phenotypic analysis offour human medulloblastoma cell lines and transplantable xenografts. JNeuropathol Exper Neurol 48:48-68, 1989). Disordered NF proteinexpression can be found in PC12 cells, derived from a ratpheochromocytoma (Baker D. L., Reddy U. R., Pleasure S., Hardy M.,Williams M., Tartaglione M., Biegel J. A., Emanuel B. S., Presti P. L.,Kreidler B., Trojanowski J. Q., Evans A., Roy A. R., VenkataakrishnanG., Chen J., Ross A. H., Pleasure D.: Human central nervous systemprimitive neuroectodermal tumor expressing NGF receptors: CHP707m. AnnNeurol 28:136-145, 1990; Trojanowski J. A., Friedman H. S., Burger P.C., Bigner D. D.: A rapidly dividing human medulloblastoma cell line(D283) expresses all 3 neurofilament subunits. Am J Pathol 126:358-363,1987). In this NGF-responsive cell line, NGF treatment enhanced theexpression of NF-L and NF-M. ATRA induced neuronal differentiation ofNT2 cells, derived from a human teratocarcinoma, includes induction ofexpression of all 3 NF constituents as the cells leave the mitotic cycleand produce neurites (ATRA induced neuronal differentiation of NT2cells, derived from a human teratocarcinoma, includes induction ofexpression of all 3 NF constituents as the cells leave the mitotic cycleand produce neurites) (Pleasure S. J., Page C., Lee V. M. -Y.: Pure,postmitotic, polarized human neurons derived from NTera2 cells provide asystem for expressing exogenous proteins in terminally differentiatedneurons. The J of Neuroscience 12:1803-1815, 1992; Lee M.-Y., Andrews P.W.: Differentiation of NTERA-2 clonal human embryonal carcinoma cellsinto neurons involves the induction of all three neurofilament proteins.The J of Neuroscience 6:514-521, 1986). This induction of NF proteinexpression is accompanied by morphological changes resembling normalneurons (Lee M.-Y., Andrews P. W.: Differentiation of NTERA-2 clonalhuman embryonal carcinoma cells into neurons involves the induction ofall three neurofilament proteins. The J of Neuroscience 6:514-521, 1986)and these cells even show action potentials (Younkin D. P., Tang C.-M.,Hardy M., Reddy U. R., Shi Q.-Y., Pleasure S. J., Lee V. M.-Y.:Inducible expression of neuronal glutamate receptor channels in the NT2human cell line. Proc Natl Acad Sci USA 90:2174-2178, 1993).

The Hu antigen is a novel neuronal differentiation antigen notpreviously studied in medulloblastoma-derived cell lines. This antigen,originally identified by antibodies in patients with paraneoplasticencephalomyelitis/sensory neuronopathy (Dalmau J., Furneaux H. M.,Gralla R. J., Kris M. G., Posner J. B.: Detection of the anti-Huantibody in the serum of patients with small cell lung cancer--Aquantitative Western blot analysis. Ann Neurol 27:544-552, 1990; SzaboA., Dalmau J., Manley G., Rosenfeld M., Wong E., Henson J., Posner J.B., Furneaux H.: HuD, a paraneoplastic encephalomyelitis antigen,contains RNA-binding domains and is homologous to Elav and Sex-lethal.Cell 67:325-333, 1991; Furneaux H. M., Reich L., Posner J. B.:Autoantibody synthesis in the central nervous system of patients withparaneoplastic syndromes. Neurol. 40:1085-1091, 1990), has been shown tobe expressed in small cell lung tumors and neurons (Dalmau J., FurneauxH. M., Gralla R. J. , Kris M. G., Posner J. B.: Detection of the anti-Huantibody in the serum of patients with small cell lung cancer--Aquantitative Western blot analysis. Ann Neurol 27:544-552, 1990). Thegene encoding the protein which reacts with anti-Hu antibodies has beensequenced and cloned (Szabo A., Dalmau J., Manley G., Rosenfeld M., WongE., Henson J., Posner J. B., Furneaux H.: HuD, a paraneoplasticencephalomyelitis antigen, contains RNA-binding domains and ishomologous to Elav and Sex-lethal. Cell 67:325-333, 1991). Theremarkable homology to the Drosophila proteins Elav and Sex-lethalsuggests a role for Hu in the maturation of mammalian neurons. It may bea neuron-specific RNA processing protein (Szabo A., Dalmau J., ManleyG., Rosenfeld M., Wong E., Henson J., Posner J. B., Furneaux H.: HuD, aparaneoplastic encephalomyelitis antigen, contains RNA-binding domainsand is homologous to Elav and Sex-lethal. Cell 67:325-333, 1991). Hu wasdetectable in both cell lines with features of neuronal differentiation,D283 and BE(2)C cells, and was found to be modulated by PA. Thisdevelopmental antigen, which is expressed at a very early stage ofneuronal commitment way be an indicated for distinct differentiationpathways and a new marker to better define the differentiation state andsubgroups of medulloblastomas.

The effect of PA administration on NF protein expression in D283 cellswas characterized by an increase of NF-L and NF-M accompanied by anincrease in the Hu antigen. ATRA induced opposite effects on NF-M and Husuggesting that ATRA does not induce differentiation of D283 cells alongthe neuronal pathway (Lieberman F., Finzi D., Ferro J.: Retinoic aciddecreases proliferation and Hu expression in medulloblastoma cells. AnnNeurol 32:286, 1992).

Hu expression did not correlate with synaptophysin expression. None ofthe medulloblastoma cell lines expressed synaptophysin before or aftertreatment. This 28kDa glycoprotein, a component of presynaptic vesicles,is characteristic of neuroendocrine cells and neuronally differentiatedtumors (Molenaar W. M., Baker D. L., Pleasure D., Trojanowski J. Q.: Theneuroendocrine and neural profile of neuroblastomas,ganglioneuroblastomas and ganglioneuromas. Am J. Pathol 136:375-382,1990). Synaptophysin has been reported in a large fraction ofmedulloblastomas (Coffin C. M., Braun J. T., Wisk M. R., et al.: aclinicopathologic and immunohistochemical analysis of 53 cases ofmedulloblastoma with emphasis on synaptophysin expression. Mod Path3:164, 1990). It is considered a marker of neuronal differentiation,although the prognostic relevance of such expression is unclear.

Also studied were the effects of PA in a neuroblastoma cell line, whichrepresents a well characterized bipotential differentiation modelcapable of differentiation towards a neuronal or a schwann/glial cellphenotype (Biedler J. L., Casals D., Chang T., Meyers M. B., Spengler B.A., Ross R. A.: Multidrug resistant human neuroblastoma cells are moredifferentiated than controls and retinoic acid further induceslineage-specific differentiation. Advances in Neuroblastoma Research3:181-191, 1991, Wiley-Liss, Inc.; Ross R. A., Bossart E., Spengler B.A., Biedler J. L.: Multipotent capacity of morphologically intermediate(I-type) human neuroblastoma cells after treatment withdifferentiation-inducing drugs. Advances in Neuroblastoma Research3:193-201, 1991, Wiley-Liss, Inc.; Ciccarone V., Spengler B. A., MeyersM. B., Biedler J. L., Ross R. A.: Phenotypic diversification in humanneuroblastoma cells: Expression of neural crest lineages. Cancer Res49:219-225, 1989; Ross Ra, Spengler B. A., Rettig W. R., Biedler J. L.:Permanent phenotypic conversion of human neuroblastoma I-type cells.Proc Am Assoc Cancer Res 35:44, 1993). These effects were studied inorder to compare the response to medulloblastoma and glioma cell lines.The cell line, BE(1)C, established by Biedler (Biedler J. L., Helson L.,Spengler B. A.: Morphology and growth, tumorigenicity of humanneuroblastoma cells in continuous culture. Cancer Res 33:2643-2652,1973), represents an intermediate type subclone with features of bothneuronal (N-) and glial/schwann (S-) type cells and can be neuronallydifferentiated by ATRA and differentiated towards a S-phenotype withBUdR and dBcAMP (Ross R. A., Bossart E., Spengler B. A., Biedler J. L.:Multipotent capacity of morphologically intermediate (I-type) humanneuroblastoma cells after treatment with differentiation-inducing drugs.Advances in Neuroblastoma Research 3:193-201, 1991, Wiley-Liss, Inc.).During ontogenesis, the appearance of NF proteins coincides with aswitch away from the expression of vimentin in precursor cells to theexpression of NF proteins in nascent neurons derived from theseprecursors (Tapscot 1981, supra). PA- and ATRA-induced growth inhibitionin BE(2)C cells was accompanied by changes in the expression of NFproteins, Hu and vimentin with PA and ATRA inducing opposite effects. Inthe BE(2)C differentiation model PA caused similar phenotypic changes asreported with BUdR and dBcAMP with differentiation towards theS-phenotype (Ross R. A., Bossart E., Spengler B. A., Biedler J. L.:Multipotent capacity of morphologically intermediate (I-type) humanneuroblastoma cells after treatment with differentiation-inducing drugs.Advances in Neuroblastoma Research 3:193-201, 1991, Wiley-Liss, Inc.).The choice of pathway appears cell line dependent, since D283differentiate in a neuronal direction with PA but BE(2)C differentiatein a schwann/glial direction. The underlying mechanisms at this switchpoint are unknown.

The mechanisms by which PA produces its effects are not understood. Thehypothesis that glutamine depletion is responsible seems unlikely. PA iseffective in rat and murine systems, in which PA conjugates glycinerather than glutamine (James M. O., Smith R. L., Williams R. T., et al.:Conjugation of phenylacetic acid in man, subhuman primates and somenon-human species. Proc R. Soc Lond 182:25-35, 1972) and PA's effect onin vitro growth of U251 human malignant astrocytoma cells could not beblocked by glutamine supplementation. These findings are in agreementwith previous data described herein on the interaction of glutamine withPA in non-neuroectodermal derived cell lines. PA appears to causehypomethylation of DNA in vitro (Anjusin B. F., Bashkite E. A.,Freidrich A. et al.: Metilirovanie DNK V prostkach psenicy Ivligjanijefytogormonor. Biochemia 46:47-53, 1981), but the significance of thisobservation is unclear. In HL-60 cells, PA treatment is associated withdecreased c-myc expression. See supra.

The transforming growth factors beta (TGFβ1, β2 and β3) are polypeptidesthat influence the proliferation and differentiation of many cell types(Sporn M. B., Roberts A. B.: Peptide growth factors and their receptorsI:419-439, 1990, Springer Verlag). As described herein, there is aadecrease in TGFβ2 mRNA levels after PA treatment of PC3 prostatecarcinoma cells. This family of growth regulatory molecules has beenshown to be important to the biology of astrocytoma growth. Bothnumerous cell lines (Falk L. A., DeBenedetti F., Lohrey N. et al.:Induction of TGFβ1 receptor expression and TGFβ1 protein production inretinoic acid treated HL-60 cells. Blood 77:1248-1255, 1991) and primarytumor tissue (Clark W. C., Bressler J.: TGFβ-like activity in tumors ofthe central nervous system. J Neurosurg 68:920-924, 1988; Samuels V.,Barett J. M., Brochman S. et al.: Immunocytochemical study oftransforming growth factor expression by benign and malignant gliomas.Am J Pathol 134:895-902, 1989) have been shown to express mRNA for oneor more of the 3 TGFβ isoforms. Malignant glioma derived cell linesdemonstrate variable proliferative responses to TGFβ in vitro (Falk L.A., DeBenedetti F., Lohrey N. et al.: Induction of TGFβ1 receptorexpression and TGFβ1 protein production in retinoic acid treated HL-60cells. Blood 77:1248-1255, 1991) and the responses may correlate withhyperdiploid or aneuploid state, and morphologic criteria of anaplasia.TGFβ appears to be a growth inhibitory factor for some astrocytoma cellsand a mitogenic factor for others. The molecular basis for the differentresponse is unclear. The role of TGFβ in the biology of medulloblastomagrowth and differentiation has not been studied, in part due to thedifficulty in establishing medulloblastoma cell lines and the relativepaucity of cases relative to malignant astrocytomas.

PA does appear to modulate TGFβ2 production by Daoy medulloblastoma aswell as PC3 prostate carcinoma cells (described supra). Increasedamounts of TGFβ2 were detected by quantitative ELISA after Daoy cellswere exposed to PA. However, the data suggest that these changes do notmediate the antiproliferative effects. Exogenous TGFβ1 and TGFβ2 had noinfluence on Daoy cell growth. Neutralizing Abs against TGFβ1 and TGFβ2did not block PA-mediated growth inhibition of U251 and Daoy cells.

The in vitro data suggest that PA and related compounds may be usefulagents in the treatment of medulloblastomas and other primary CNStumors. These agents warrant further studies in animal systems and inhuman clinical trials.

To define the capability of PA to inhibit growth and inducedifferentiation in neuroectodermal tumor cell lines, the effects of PAin 1 human (U251) and 2 rodent (C6, RG2) cell lines of glial origin aswell as 2 human medulloblastoma cell lines (Daoy, D283) were studied.The effects of PA on these cell lines were compared to the humanneuroblastoma cell line BE(2)C since this cell line represents a wellcharacterized bidirectional differentiation model for neuronal andschwann/glial cell differentiation.

Section Q: NaPA and NaPB Human Studies

The growth inhibition and differentiating effects of sodiumphenylacetate against hematopoietic and solid tumor cell lines hasaroused clinical interest in its use as an anticancer drug. Thenon-linear pharmacokinetics, metabolism, toxicity, and clinical activityof PA when administered by continuous i.v. infusion (CIVI) for 2 weekshave-recently been described. In this phase I, PA was administered i.v.twice daily (BID) in an attempt to minimize drug accumulation whilemaximizing peak serum drug concentrations. Twenty-seven cycles oftherapy were given to 18 patients at two dose levels (125 and 150 mg/kgBID for 14 days). Detailed pharmacokinetic studies in 8 patientsconclusively showed that PA induces its own clearance by a factor of 44%over two weeks. Dose-limiting toxicity consisted of reversible centralnervous system depression associated with nausea and hypoacusis.Clinical improvement was observed in eight patients. Three of sevenpatients with refractory malignant glioma had improved performancestatus for up to 9 months. One had a partial response and another aminor response on MRI. Improvement was also seen in five of ninepatients with hormone-independent prostate cancer (HIPC). One had agreater than 50% decline in PSA, one had resolution of disseminatedintravascular coagulation and one noted decreased bone pain that lastedfor more than 1 month. Two patients maintained an improved performancestatus for more than 8 months, one of whom demonstrated healing ofblastic bone metastases. These results suggest that PA has activity inmalignant gliomas and HIPC. The recommended schedule for phase IItesting is 125 mg/kg BID (one hour infusion) given in monthly cycles of14 days duration.

A first phase I trial of phenylacetate has been described above. In thattrial, the drug was administered by CIVI in an attempt to maintain drugconcentrations in the range associated with preclinical activity. Underthese conditions, phenylacetate displayed saturable kinetics andevidence for induction of its own metabolism (pharmacokineticparameters, mean±SD: Km=105.1±44.5 μg/ml, V_(max) =24.1±5.2 mg/kg/hr,Vd=19.2±3.3 L., and IR=0.0028±0.003 h⁻¹). Clinical improvement was notedin several patients with metastatic hormone-independent prostate cancerand malignant glioma who achieved serum phenylacetate concentrations of1-2 mM. Infusion rates close to the V_(max) of the metabolizing enzymewere required, however, in order to achieve concentrations of 3 mM ormore. This often resulted in rapid drug accumulation, associated withneurological toxicity once phenylacetate levels exceeded 6 mM. Theselimitations led to the investigation of a different dosing schedulewherein the drug was given as a one-hour infusion twice daily, based onthe assumption that elevations in phenylacetate concentrations might bewell tolerated if transient, and that intermittent dosing might helpcontrol drug accumulation.

Example 29

In vivo trials with NaPA B.I.D.

Patient Population. Adults with advanced solid tumors refractory toconventional therapy, a performance status greater than 60% onKamofsky's scale, normal hepatic transaminases and bilirubin, a serumcreatinine less than 1.5 mg/dl, and normal leucocyte and platelet countswere eligible for this study. The clinical protocol was reviewed andapproved by the NCI Institutional Review Board and all patients gavewritten informed consent prior to participating in the study. Eighteenpatients, 15 men and 3 women, with a mean age of 54 years (range, 32 to76) were enrolled between July and October 1993. Disease distributionincluded metastatic, hormone-independent prostate cancer (9 patients),primary CNS tumors (7 patients), renal cell cancer (1 patient) andsarcoma (1 patient). Four patients, two with gliomas and two withprostate cancer, had received prior treatment with phenylacetate givenby CIVI.

Drug Preparation and Administration. Sodium phenylacetate for injection(Elan Pharmaceutical Research Co., Gainesville, Ga.) was prepared fromsterile sodium phenylacetate powder by the Pharmacy Department of theClinical Center, NIH, in vials containing a drug concentration of 500mg/ml in sterile water for injection, USP. Sodium hydroxide orhydrochloric acid was added to adjust the final pH to 7.4. Doses ofsodium phenylacetate to be infused over 1 hour were prepared in 250 mlof sterile water for injection, USP, and were administered using aninfusion pump. The experiment used the one compartment, non-linearpharmacokinetic model and population parameters derived from previousexperience with phenylacetate to stimulate the course of severalintermittent dosing regimens. The primary objective was to design onethat would expose most patients to transient phenylacetateconcentrations in excess of 3 mM and maintain trough concentrations ofapproximately 2 mM. These appeared necessary for antitumor activity invitro and had been well tolerated by patients for as long as 2 weeks.The secondary objective was to pharmacokinetically determine the optimaldose of phenylacetate to achieve the above and minimize the number ofescalation steps in the trial by beginning at that dose.

Phenylacetate was delivered at two dose levels: ten patients weretreated at 125 and eight at 150 mg/kg/dose, BID, for 14 consecutivedays. Cycles of therapy were repeated every 4 weeks. The dose wasincreased in sequential cohorts of at least three patients. Individualpatients could escalate from one dose level to the next with sequentialcycles provided they had experienced no drug-related toxicity and theirdisease was stable or improved.

Sampling Schedule. Serum drug concentrations were measured twice a dayin all patients, immediately prior to and 15 minutes following theadministration of the 05:00 PM infusion. To assess the possibility thatphenylacetate induces its own clearance, 8 patients underwent moreintensive drug level monitoring on days 1, 2 or 3 and days 12, 13 or 14of the two weeks of therapy. In these patients, blood was also obtainedat 0, 65, 90, 105, 120, 150, 180, 210, 240, 300 and 360 minutes from thebeginning of the 08:00 AM infusion. This allowed for a comparison to bemade between AUCs generated from identical doses of phenylacetate at thebeginning and at the end of therapy, any difference reflecting a changein drug clearance over this period of time.

Analytical Method. Blood was drawn into plain glass Vacutainer® tubesand refrigerated. Serum was separated and frozen at -85° C. within 12hours of collection. The HPLC method for measuring serum concentrationsof phenylacetate and phenylacetylglutamine is described elsewhereherein. Briefly, 100 μl of 10% perchloric acid were added to 200 μl ofserum to precipitate the proteins. After centrifugation, the supernatantwas neutralized with 25 μl of a 20% solution of potassium bicarbonate.Following a second centrifugation, 20 μl of supernatant were injectedonto a 300×3.4 mm C-18 column heated at 60° C. Elution was performedwith an increasing gradient of acetonitrile in water from 5% to 30% over20 minutes. Its progress was followed by monitoring UV absorbance at 208nm. Characteristic elution times for phenylacetate andphenylacetylglutamine under these conditions were 17.1 and 9.8 minutes,respectively.

Determination of Responses to Treatment. Patients were seen at leastmonthly by a physician at the NCI. The response status of malignanciesother than prostate cancer and primary CNS-tumors was determined priorto each cycle of therapy, using conventional anatomic criteria. Forprostate cancer patients, criteria from the NPCP trials and publishedcriteria of PSA decline were used. A technetium bone scan was obtainedevery three months if initially positive or in the presence of new bonesymptoms. The assessment of patients with gliomas is complicated by thefrequent microscopic multicentricity of the tumor, the variability intumor-associated edema and its response to steroid therapy, andtechnical factors which precluded using the intensity of gadoliniumenhancement on MRI to determine tumor response. For these reasons,special attention was paid to changes in performance status and steroidrequirements, which were assessed at each visit. Complete response wasdefined as complete disappearance of lesions on MRI (assessment done intwo different planes), and weaning from steroids. Partial response wasdefined by conventional anatomic criteria, absence of deterioration inperformance status and stable or decreased corticosteroid requirements.Minor response was defined similarly, using 25% as the minimal limit ofsize reduction. Progressive disease was defined either by anatomiccriteria, deterioration in performance status by at least 20 points onKarnofsky's scale or the need for increasing steroid doses in order tomaintain function. Disease stabilization was defined as the absence of asignificant (more than 25%) increase or decrease in tumor size while thepatient maintained or improved his performance status at hispre-treatment level. To be scored as significant, disease stabilizationin these patients had to be maintained for at least three months.

Statistical Methods. To determine whether phenylacetate induces its ownclearance, the AUCs following a single dose of the drug at the beginningand end of PA therapy in 8 patients were compared using the Wilcoxonsigned rank test for paired data.

Pharmacokinetic Simulation and Clinical Findings. Several intermittentdosing schedules were modeled using the pharmacokinetic parametersderived from a previous trial of phenylacetate. The pharacokineticcourse of a 70 kg man given phenylacetate at 125 mg/kg/dose BID (08:00AM and 05:00 PM) was modeled. The simulation predicted peak levelsbetween 1.5 and 3.5 mM with trough concentrations below 1 mM and no drugaccumulation (95% confidence intervals).

Analysis of drug levels in 18 patients shows peak serum concentrations(mean±SD) of 3.0±1.2 mM (n=10) and 3.7±0.8 mM (n=8) at the two doselevels. Corresponding trough concentrations were 0.2±0.2 mM and 0.7±0.5mM, respectively. On average, patients spent 40% of their treatment timeabove a concentration of 2 mM. Drug accumulation associated withneurologic toxicity (highest phenylacetate concentration: 7.3 mM)occurred in a single patient treated at the second dose level.Population pharmacokinetic parameters were determined by fitting a onecompartment non-linear model to each patient's dosing and drugconcentration data, using as initial parameter estimates of the meanparameter values described elsewhere above. The pharmacokineticparameters of each individual patient were then averaged and expressedas mean parameter values with associated standard deviation:

    Km=112±34 μg/ml,

    V.sub.max =25.8±5.0 mg/kg/hr, and

    Vd=21.4±4.6 L.

Phenylacetate Clearance

The molar excretion of phenylacetylglutamine was determined from 24 hoururine collections. It accounted for 76±15% (mean±SD., n=24) of the doseof phenylacetate given over the same period of time. The recovery offree, non-metabolized drug was 3±1% of the total administered dose.

Induction of Phenylacetate Clearance. The hypothesis that phenylacetateinduces its own clearance was tested by comparing AUCs following the AMinfusion of phenylacetate at the beginning and at the end of therapy in8 patients. All exhibited a decrease in AUC, with a mean value of 44±27%(p value=0.008) between the two ends of each cycle of therapy.

Clinical Assessment of Antitumor Activity. Three of seven patients withbrain tumors displayed evidence of antitumor activity or diseasestabilization. One had a partial response accompanied by subjectiveimprovement in short-term memory. This response was confirmed byradiographic evidence of healing osteoblastic metastases (6 monthsfollow up) despite rising serum PSA concentrations over the same periodof time in a 57 year old man with metastatic, hormone-independentprostate cancer. She was also able to reduce her daily corticosteroiddose by 50%, from 8 to 4 mg of dexamethasone. Two patients, whoinitially presented with confusion, had received phenylacetate by CIVIprior to being treated on the intermittent schedule. One had a minorresponse documented radiologically after the change in regimen, whiletumor size remained stable in the second. Both maintained a 20 and 30%improvement in performance status for six and seven months,respectively. Their dose of corticosterioids was held constantthroughout their treatment.

Five of nine patients with hormone-independent prostate cancer showedclinical improvement. One experienced a greater than 50% decline in PSAsustained for a month and has maintained a performance status of 100% onKarnofsky's scale for more than five months. One with tumor-associateddisseminated intravascular coagulation (prolonged prothrombin time, lowfibrinogen, normal platelet count) stopped taking flutamide concurrentlywith starting phenylacetate. His hematological parameters normalizedduring the first week of therapy while his performance status improvedby 30%, due to reduction of bone pain. Two who had already been treatedwith phenylacetate given by CIVI with an associated improvement in bonepain and performance status, have maintained if for five additionalmonths. One of them also had evidence of healing osteoblastic metastaseson CT scan. Comparison of pre-treatment gadolinium-enhanced brain MRI ina patient with recurrent glioblastoma multiforme verus post-treatmentgadolinium-enhanced MRI after 3 cycles of phenylacetate (150 mg/kg BID),confirmed this partial response. This evidence of healing metastasescontinued despite steadily rising serum PSA concentrations over the sameperiod of time from 1.0 to 80 ng/ml. Finally, one patient has hadsubjective amelioration of bone pain for 1 month.

Clinical Toxicities. Reversible neurological toxicity was the mostcommon side effect associated with the administration of phenylacetatetwice daily. All patients experienced mild (grade I) somnolence (peakserum concentration, mean±SD: 2.9±0.5 mM). Three patients who received150 mg/kg BID developed dose-limiting neurotoxicity (serumconcentrations, mean±SD: 5.3±1.7 mM). One with glioblastoma multiformeexperienced transient spatial disorientation and hypoacusis, associatedwith drug accumulation (phenylacetate concentration: 7.3 mM). Anotherwith anaplastic astrocytoma experienced grade II somnolence that wastemporarily related to peak concentrations of phenylacetate (mean peakconcentration: 4.7 mM). A third patient with prostate cancer whosuffered from suramin-induced sensory neuropathy experienced gradualdeterioration of his condition over the first ten days of phenylacetateadministration (peak and trough levels, mean ±SD: 3.9±0.4 and 0.9±0.4mM), at which time therapy was discontinued. In this patient, the sideeffects improved gradually over 3 months.

Three patients with a history of angina pectoris, supraventriculartachycardia or palpitations associated with mitral valve prolapsereported reversible exacerbation of their usual symptoms during theinfusion of phenylacetate. This was likely related to significant fluidshifts induced by the high sodium content of the drug formulation. Nosuch symptoms were noted in patients free of cardiovascular impairment.

This trial was designed to overcome the problem of rapid drugaccumulation associated with the delivery of high doses of phenylacetateby CIVI. The goal was to achieve transient peak phenylacetateconcentrations in the range found to be active preclinically (≧2 mM) andallow enough time for drug elimination between each dose ofphenylacetate. The pharmacokinetic information derived from the firsttrial of phenylacetate was used to stimulate several intermittent dosingregimen. Administering 125 mg/kg/dose of phenylacetate twice daily (9hours apart) as 60 minute infusions, was predicted to achieve serum drugconcentrations between 3 and 5 mM without drug accumulation, in morethan 95% of patients. This dose was therefore chosen as the startingpoint for the trial.

The results confirm that most patients treated with 125 and 150mg/kg/dose of phenylacetate achieve peak serum drug concentrations inthe range of 3-5 mM. No patient treated at the first dose levelexperienced undesirable drug accumulation, which occurred in 1 of 8patients treated at the higher level. Patients' exposure to potentialactive concentrations of phenylacetate was equal to 40% of their totaltreatment time. The accuracy of the pharmacokinetic simulationsuccessfully eliminated the need for multiple escalation steps andallowed the clinical questions to be answered rapidly.

The trial enabled the characterization of the toxicity of phenylacetatewith respect to peak drug levels. The 125 mg/kg dose level wasassociated with a mean peak serum concentration of 3.0 mM and grade Ineurocortical toxicity (somnolence). The temporal relationship betweendrug infusion and the onset of somnolence was noted in all patientstreated at this dose level. Gradual recovery between each infusion wasthe rule. The second dose level (150 mg/kg BID) was associated withgrade I neurotoxicity in five patients (mean peak serum concentration:3.7 mM) and more severe toxicity in three patients whose mean peak drugconcentration was 5.3 mM. Except for the deterioration seen in a patientwith pre-existing suramin-induced sensory neuropathy, neurotoxicity fromphenylacetate has been acute and reversible.

Evidence for the induction of phenylacetate clearance is available fromthe first trial of phenylacetate. The demonstration was indirect,however, due to frequent adjustments in drug dosage within each cycle oftherapy. With the current fixed-dosing regimen, a 44% mean decline inthe AUC associated with identical doses of drug given at the beginningand end of the two week treatment was shown. This verifies thehypothesis that PA induces its own clearance by the hepatic enzymephenylacetyl Coenzyme A:glutamine acyltransferase. The induction ofmetabolism is not sustained after treatment is discontinued, lendingfurther support to the hypothesis that metabolism of phenylacetate isself-induced. The observation of autoinduction of clearance is relevantto the optimal duration of phenylacetate therapy and the eventual needfor dose modification over time.

The partial and minor responses noted in patients with malignant gliomaswere not expected from a differentiating agent causing cytostasis invitro. There is evidence (described herein), however, that phenylacetateinhibits the mevalonate pathway of cholesterol synthesis, whichmalignant astroglia rely on predominantly for their growth. This couldresult in cell death and decreased tumor size. An alternative mechanismrelates to the depletion of circulating glutamine, a major source ofenergy for various tumor cell types. The metabolism of phenylacetateyields phenylacetylglutamine and causes significant reductions of plasmaglutamine after rapid infusion. In this respect, although repeatedadministration of phenylacetate was not associated with sustaineddeclines in plasma glutamine concentrations, a 70 kg patient receiving125 mg/kg/dose twice daily would excrete more than 90 moles of glutaminea day under as urinary phenylacetylglutamine. At present, whether thisis reflected at the tumor site is not known. Assessing tumor response inpatients treated with differentiating agents may be problematic. Thedifficulty is especially acute in patients with hormone-independentprostate cancer, for whom PSA has been proposed as the best follow uptool available. Since PSA production is organ-specific and directlycorrelated with the degree of tumor differentiatio, phenotypic reversioninduced by phenylacetate could potentially be associated with risingserum levels of the marker. This would render PSA clinically useless asa marker of disease burden and emphasize the role of the moretraditional anatomic criteria. The latter are unfortunatelyinappropriate for evaluating the vast majority of patients with advancedprostate cancer who lack soft tissue metastases. Therefore, conventionalanatomic criteria and performance status scores (including pain relief)be used to describe responses until further experience with PSA in thiscontext becomes available. Applying these guidelines, five of ninepatients with metastatic, hormone-independent prostate cancer appear tohave clinically benefitted from phenylacetate therapy.

Phenylacetate given at a dose of 125 mg/kg BID for two consecutive weeksis well tolerated and is associated with antitumor activity in patientswith high grade gliomas and advanced prostate cancer.

In vivo, PA is conjugated with glutamine to form phenylacetylglutamine(PAG). PA, however, has an unpleasant odor which limits patientacceptability. Phenylbutyrate (PB) is an odorless compound which alsopossesses in vitro activity (0.5 to 2 mM) and is believed to undergorapid conversion to PA by in vivo β-oxidation.

A phase I study was undertaken to examine the pharmacokinetics (PK) ofPB and characterized the disposition of the two metabolites (PA andPAG). Fourteen patients with cancer (mean age 51.8±13.8 years) receiveda 30 min. infusion of PB at three dose levels (600, 1200 and 2000mg/m²). Serial blood samples and a 24 hour urine collection wereobtained. Samples were assayed by HPLC (CV<10%). A model tosimultaneously describe the PK of all three compounds was developedusing ADAPT II. Data were modeled as molar equivalents.

The model fit the data well as demonstrated by mean (±S.D.) coefficientsof determination (r²) for PB, PA, and PAG which were 0.96±0.07,0.88±0.10 and 0.92±0.06, respectively. The intrapatient CV % around theparameter estimates were small (range 87.2% to 33.5%). PB achieved peakconcentration in the range of in vitro tumor activity and exhibitedsaturable elimination (Km=34.1±18.1 μg/ml and V_(max) =18.1±18mg/hr/kg). Metabolism was rapid; T_(max) for PA and PAG was 1 and 2hours, respectively. The conversion of PB to PA was extensive(80±12.6%), but serum concentration of PA were low due to rapid,subsequent conversion to PAG. The ratio of PB AUC/PA AUC was 2.25. PB,thus, shows activity as an independent therapeutic agent, notnecessarily solely as a prodrug of PA.

Section R: NaPA and NaPB Alterations of Lipid Metabolism

NaPA has shown consistent ability to induce biochemical changes relatedto lipid metabolism. Such activity is of value in the treatment of lipidmetabolism related disorders, including cancer and cardiovasculardisease.

As discussed previously, NaPA and its derivatives activate the humanperoxisomal proliferator activated receptor (PPAR). The PPAR regulatesthe expression of key enzymes involved in lipid metabolism such asacyl-CoA oxidase and the cytochrome P450IV family of enzymes. Otheragents are capable of activating the PPAR, e.g., fatty acids andhypolipidimic drugs such as fibric acid derivatives (clofibrate). Drugsin this group have been highly effective in lowering serum triglyceridelevels and therefore are widely used to reduce the level of atherogeniclipoproteins associated with elevated risk of coronary artery disease.

Glioma cells accumulate lipids following treatment with PA. Thus, cancercells are dependent on free fatty acids for some energy metabolism.There is a 30-50% reduction is lipids in patients treated with PA andPB. The lipid-lowering capacity of PA is demonstrated in several cancerpatients treated with 300-350 mg/kg/day (IV) NaPA. Table 29. Thesepatients showed a 30-60% decline in triglycerides (TG) levels while onthe therapy.

For example: Patient S. L. showed TG levels reduced from 169±5 mg/dl to75±21 mg/dl during a course of 14 IV treatments with 350 mg/kg/day PA.Two weeks after PA treatment was discontinued, TG levels recovered and 5reached 185 mg/dl. Similar changes were observed in other patients. SeeTable 29

                  TABLE 29    ______________________________________    Serum Triglyceride Levels Upon Treatment with PA    Patient        TG Before                            TG After    ______________________________________    1. S. L.       169       75    2. S. G.       260      144    3. H. L.       295      135    4. L. A.       228      143    ______________________________________

It thus appears that PA and PB stimulate PPAR (increase of peroxidationand decrease of DNA synthesis). In addition, because PA/PB also inhibitsthe mevalonate pathway (see section B. above) which decreases proteinprenylation (such as ras protein, G proteins and nuclear lamins), PAacts to decrease cholesterol synthesis prior to DNA synthesis. A reducedprenylated ras fails to activate a proliferation signal. Thus, as notedabove, HMG CoA reductase inhibitors, e.g. lovastatin, provide asynergistic combination with PA/PB as measured by invasiveness andmetastasis.

Furthermore, the TG-reducing activity of PA is essentially free ofadverse effects, and is as effective or better than that of clofibrateand its derivatives (Olsson et al., Atherosclerosis 27:279-297, 1977).

Section S: Modes of Drug Administration

NaPA (or PAA derivatives) may be administered locally or systemically.Systemic administration means any mode or route of administration whichresults in effective levels of active ingredient appearing in the bloodor at a site remote from the site of administration of said activeingredient.

The pharmaceutical formulation for systemic administration according tothe invention may be formulated for intravenous, intramuscular,subcutaneous, oral, nasal, enteral, parenteral, intravesicle or topicaladministration. In some cases, a combination of types of formulationsmay be used simultaneously to achieve systemic administration of theactive ingredient.

Suitable formulations for oral administration include hard or softgelatin capsules, dragees, pills, tablets (including coated tablets),elixirs, suspensions, and syrups or inhalations.

Solid dosage forms in addition to those formulated for oraladministration include rectal suppositories.

The compounds of the present invention may also be administered in theform of an implant.

Suitable formulations for topical administration include creams, gels,jellies, mucilages, pastes and ointments.

Suitable injectable solutions include intravenous, subcutaneous, andintramuscular injectable solutions. The compounds of the presentinvention may also be administered in the form of an infusion solutionor as a nasal inhalation or spray.

The compounds of the present invention may also be used concomitantly orin combination with selected biological response modifiers, e.g.,interferons, interleukins, tumor necrosis factor, glutamine antagonists,hormones, vitamins, as well as anti-tumor agents and hematopoieticgrowth factors, discussed above.

It has been observed that NaPA is somewhat malodorous. Therefore, it maybe preferable to administer this compound in the presence of any of thepharmaceutically acceptable odor-masking excipients or as its precursorphenylbutyrate (or a derivative or analog thereof) which has nooffensive odor.

The PAA and its pharmaceutically acceptable derivatives to be used asantitumor agents can be prepared easily using pharmaceutical materialswhich themselves are available in the art and can be prepared byestablished procedures. The following preparations are illustrative ofthe preparation of the dosage forms of the present invention, and arenot to be construed as a limitation thereof.

Example 30

Parenteral Solution 1

A sterile aqueous solution for parenteral administration containing 200mg/ml of NaPA for treating a neoplastic disease is prepared bydissolving 200 g. of sterilized, micronized NaPA in sterilized NormalSaline Solution, qs to 1000 ml. The resulting sterile solution is placedinto sterile vials and sealed. The above solution can be used to treatmalignant conditions at a dosage range of from about 100 mg/kg/day toabout 1000 mg/kg/day. Infusion can be continuous over a 24 hour period.

Example 31

Parenteral Solution 2

A sterile aqueous solution for parenteral administration containing 50mg/ml of NaPA is prepared as follows:

    ______________________________________    Ingredients      Amount    ______________________________________    NaPA, micronized 50          g.    Benzyl alcohol   0.90%       w/v    Sodium chloride  0.260%      w/v    Water for injection, qs                     1000        ml    ______________________________________

The above ingredients, except NaPA, are dissolved in water andsterilized. Sterilized NaPA is then added to the sterile solution andthe resulting solution is placed into sterile vials and sealed. Theabove solution can be used to treat a malignant condition byadministering the above solution intravenously at a flow rate to fallwithin the dosage range set forth in Example 30.

Example 32

Parenteral Solution 3

A sterile aqueous solution for parenteral administration containing 500mg/ml of sodium phenylbutyrate is prepared as follows:

    ______________________________________    Ingredients      Amount    ______________________________________    Sodium phenylbutyrate                     500         g.    Dextrose         0.45%       w/v    Phenylmercuric nitrate                     0.002%      w/v    Water for injection, qs                     1900        ml.    ______________________________________

The preparation of the above solution is similar to that described inExamples 30 and 31.

Example 33

Tablet Formulation 1

A tablet for oral administration containing 300 mg of NaPA is preparedas follows:

    ______________________________________    Ingredients      Amount    ______________________________________    NaPA             3000         g.    Polyvinylpyrrolidone                     225          g.    Lactose          617.5        g    Stearic acid     90           g.    Talc             135          g.    Corn starch      432.5        g.    Alcohol          45           L    ______________________________________

NaPA, polyvinylpyrrolidone and lactose are blended together and passedthrough a 40-mesh screen. The alcohol is added slowly and thegranulation is kneaded well. The wet mass is screened through a 4-meshscreen, dried overnight at 50° C. and screened through a 20-mesh screen.The stearic acid, talc and corn starch is bolted through 60-mesh screenprior to mixing by tubing with the granulation. The resultinggranulation is compressed into tablets using a standard 7/16 inchconcave punch.

Example 34

Tablet Formulation 2

A tablet for oral administration containing 200 mg of sodiumphenylbutyrate is prepared as follows:

    ______________________________________    Ingredients        Amount    ______________________________________    Sodium phenylbutyrate                       2240        g.    Compressible sugar (Di-Pac)                       934         g.    Sterotex           78          g.    Silica gel (Syloid)                       28          g.    ______________________________________

The above ingredients are blended in a twin-shell blender for 15 minutesand compressed on a 13/22 inch concave punch.

Example 35

Intranasal Suspension

A 500 ml sterile aqueous suspension is prepared for intranasalinstallation as follows:

    ______________________________________    Ingredients      Amount    ______________________________________    NaPA, micronized 30.0        g.    Polysorbate 80   2.5         g.    Methylparaben    1.25        g.    Propylparaben    0.09        g.    Deionized water, qs                     500         ml    ______________________________________

The above ingredients, with the exception of NaPA, are dissolved inwater and sterilized by filtration. Sterilized NaPA is added to thesterile solution and the final suspensions are aseptically filled intosterile containers.

Example 36

Ointment

An ointment is prepared from the following ingredients:

    ______________________________________    Ingredients     Amount    ______________________________________    NaPA            10           g.    Stearyl alcohol 4            g.    White wax       8            g.    White petrolatum                    78           g.    ______________________________________

The stearyl alcohol, white wax and white petrolatum are melted over asteam bath and allowed to cool. The NaPA is added slowly to the ointmentbase with stirring.

Example 37

Lotion

    ______________________________________    Ingredient              Amount    ______________________________________    Sodium phenylbutyrate   1.00     g.    Stearyl methylcellulose (4,500) Solution (2%)                            25.00    ml    Benzalkonium chloride   0.03     g.    Sterile water           250.00   ml    ______________________________________

The benzalkonium chloride is dissolved in about 10 ml. of sterile water.The sodium phenylbutyrate is dispersed into methylcellulose solution bymeans of vigorous stirring. The methylcellulose (4,500) used is a highviscosity grade. The solution of benzalkonium chloride is then addedslowly while stirring is continued. The lotion is then brought up to thedesired volume with the remaining water. Preparation of the lotion iscarried out under aseptic conditions.

Example 38

Dusting Powder

    ______________________________________    Ingredients                Amount    ______________________________________    NaPA                       25 g.    Sterilized absorbable maize starch BP dusting powder                               25 g.    ______________________________________

The dusting powder is formulated by gradually adding the sterilizedabsorbable dusting powder to NaPA to form a uniform blend. The powder isthen sterilized in conventional manner.

Example 39

Suppository, Rectal and Vaginal Pharmaceutical Preparations

Suppositories, each weighing 2.5 g. and containing 100 mg. of NaPA areprepared as follows:

    ______________________________________    Ingredients        Amount/1000    ______________________________________    Suppositories NaPA, micronized                       100         g.    Propylene glycol   150         g.    Polyethylene glycol 4000, qs                       2500        g.    ______________________________________

NaPA is finely divided by means of an air micronizer and added to thepropylene glycol and the mixture is passed through a colloid mill untiluniformly dispersed. The polyethylene glycol is melted and the propyleneglycol dispersion added slowly with stirring. The suspension is pouredinto unchilled molds at 40° C. Composition is allowed to cool andsolidify and then removed from the mold and each suppository is foilwrapped.

The foregoing suppositories are inserted rectally or vaginally fortreating neoplastic disease.

Example 40

Intravesical Treatment

Local application of PA/PB or a derivative/analog to a body surface,such as a mucosal surface, or within a body cavity, such as the bladder,kidney, uterine, vagina etc., can be used to prevent or treat apathology involving that surface or cavity. In this manner, the PA canbe targeted selectively to the particular surface or cavity so as tomaintain a relatively high concentration of the PA at that site comparedto elsewhere in the body.

For instance, bladder cancer can be treated by an intravesical method.The bladder cancer patient can abstain from food and water for asubstantial period of time prior to the treatment, such as for 8-12hours. Following this abstention, the patient can be catheterized.Approximately 50-150 mls of a solution of having a concentration ofapproximately 2.0-200.0 mM, preferably 2-20 mM, sodium phenylacetate or1.0-100 mM, preferably 1-10 mM, sodium phenybutyrate or other equipotentamount of a PA analog will be instilled directly into the bladder. Thepatient will then be requested to retain the instilled fluid for as longas possible. This treatment will be repeated, such as once daily fortwo-four weeks followed by two weeks of drug holiday. Cycles such asthis can be repeated, such as for up to 6 months. Similarly, kidneycancers could also be similarly treated.

It is known that intracellular glutathione plays a major role indetoxification and repair of cellular injury by chemical and physicalcarcinogens. NaPA treatment of normal or tumor cells markedly inducedthe activity of intracellular glutathione approximately 2-10 folddepending on growth conditions. Nontoxic agents that can induceglutathione are highly desirable since these are likely to protect cellsfrom damage by a variety of chemical carcinogens and ionizing radiation.

Taken together, the present invention demonstrates that NaPA, NaPB andother PAA derivatives have valuable potential in cancer prevention incase such as high risk individuals, for example, heavy smokers withfamilial history of lung cancer, inherited disorders of concogeneabnormalities (Li-Fraumeni syndrome), individuals exposed to radiation,and patients in remission with residual disease. Furthermore, thesecompounds can be used in combination with other therapeutic agents, suchas chemicals and radiation, to enhance tumor responses and minimizeadverse effects such as cytotoxicity and carcinogenesis. The antitumoractivity, lack of toxicity, and easy administration qualify NaPA as apreferred chemopreventive drug.

What is claimed is:
 1. A method of treating a neoplastic condition in asubject comprising administering a therapeutic amount of a retinoid incombination with a therapeutic amount of a compound of the formula:##STR5## wherein R₀ is aryl, phenoxy, substituted aryl or substitutedphenoxy;R₁ and R₂ are, independently, H, hydroxy, lower alkoxy, lowerstraight or branched chain alkyl or halogen; R₃ and R₄ are,independently, H, hydroxy, lower alkoxy, lower straight or branchedchain alkyl or halogen; and n is an integer from 0 to 2; apharmaceutically-acceptable salt thereof or a mixture thereof.
 2. Themethod of claim 1, wherein the retinoid is all-trans-retinoic acid. 3.The method of claim 1, wherein the retinoid is 9-cis-retinoic acid. 4.The method of claim 1, wherein the neoplastic condition isneuroblastoma.
 5. The method of claim 1, wherein the compound is sodiumphenylacetate.
 6. The method of claim 1, wherein the compound is sodiumphenylbutyrate.
 7. The method of claim 1, wherein the therapeutic amountof the compound is from 50 to 1000 mg/kg/day.
 8. The method of claim 1,wherein the therapeutic amount of the compound is from 300 to 500mg/kg/day.
 9. The method of claim 1, wherein the therapeutic amount ofthe compound is from 150 to 250 mg/kg/day.
 10. The method of claim 1,wherein R₀ is aryl or phenoxy, the aryl and phenoxy being unsubstitutedor substituted with, independently, one or more halogen, hydroxy orlower alkyl.
 11. The method of claim 1, whereinR₀ is phenyl, naphthyl,or phenoxy, the phenyl, naphthyl and phenoxy being unsubstituted orsubstituted with, independently, one or more moieties of halogen,hydroxy or lower alkyl.
 12. The method of claim 1, whereinR₀ is phenyl,naphthyl, or phenoxy, the phenyl, naphthyl and phenoxy beingunsubstituted or substituted with, independently, halogen, hydroxy orlower alkyl of from 1 to 4 carbon atoms; R₁ and R₂ are, independently,H, hydroxy, lower alkoxy of from 1 to 2 carbon atoms, lower straight orbranched chain alkyl of from 1 to 4 carbon atoms or halogen; and R₃ andR₄ are, independently, H, lower alkoxy of from 1 to 2 carbon atoms,lower straight or branched chain alkyl of from 1 to 4 carbon atoms orhalogen.
 13. The method of claim 1, wherein n is 0; R₀ is aryl orsubstituted aryl; R₁ and R₂ are H, lower alkoxy, or lower alkyl;pharmaceutically-acceptable salts thereof, or mixtures thereof.
 14. Themethod of claim 1, wherein the compound is α-methylphenylacetic acid,α-ethylphenylacetic acid, α-hydroxyphenylacetic acid,α-methoxyphenylacetic acid, 1-naphthylacetic acid, 4-chlorophenylaceticacid, 4-iodophenylacetic acid, 4-fluorophenylacetic acid,3-chlorophenylacetic acid, 2-chlorophenylacetic acid,2,6-dichlorophenylacetic acid, 2-methylphenylacetic acid,3-methylphenylacetic acid, 4-methylphenylacetic acid, phenoxypropionicacid, 4-chlorophenylbutyric acid, 4-iodophenylbutyric acid,4-fluorophenylbutyric acid, 3-chlorophenylbutyric acid, or2-chlorophenylbutyric acid.
 15. The method of claim 1, wherein thecomposition is administered topically.
 16. The method of claim 15,wherein the compound of the composition is applied at a concentration offrom about 0.1 mM to about 10 mM.
 17. The method of claim 1, wherein thecomposition is administered ocularly.
 18. The method of claim 1, whereinthe composition is administered orally.
 19. The method of claim 1,wherein the composition is administered in the form of a suppository.20. The method of claim 1, wherein the composition is administeredparenterally.
 21. The method of claim 1, wherein the composition isadministered intermittently.
 22. The method of claim 1, wherein thecomposition is administered continuously.
 23. The method of claim 1,wherein the composition is administered intravesically.
 24. The methodof claim 1, wherein the neoplastic condition is neuroblastoma,myelodysplasia, non-small cell lung cancer, prostatic carcinoma,melanoma, Kaposi's sarcoma, lymphoma, leukemia, adenocarcinoma, breastcancer, osteosarcoma, fibrosarcoma, squamous cancer, malignant glioma,non-malignant glioma, benign prostatic hyperplasia, papillomavirusinfection, bladder carcinoma, kidney cancer, astrocytoma, mesothelioma,medulloblastoma, Lennert's T-Cell lymphoma, Burkitt's lymphoma,Hodgkin's lymphoma, colon carcinoma, nasopharyngeal carcinoma,rhabdomyosarcoma, or multiple myeloma.